U.S. patent application number 16/803937 was filed with the patent office on 2020-10-08 for delivery of biomolecules to pbmcs to modify an immune response.
The applicant listed for this patent is SQZ Biotechnologies Company. Invention is credited to Howard BERNSTEIN, Katarina BLAGOVIC, Matthew BOOTY, Kelan HLAVATY, Scott LOUGHHEAD, Emrah Ilker OZAY, Armon R. SHAREI, Carolyne Kelly SMITH, Defne YARAR.
Application Number | 20200318066 16/803937 |
Document ID | / |
Family ID | 1000004958141 |
Filed Date | 2020-10-08 |
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United States Patent
Application |
20200318066 |
Kind Code |
A1 |
SHAREI; Armon R. ; et
al. |
October 8, 2020 |
DELIVERY OF BIOMOLECULES TO PBMCs TO MODIFY AN IMMUNE RESPONSE
Abstract
The present application provides peripheral blood mononuclear
cells comprising an antigen, methods of manufacturing such PBMCs,
and methods of using such PBMCs, such as for modulating an immune
response in an individual. In some embodiments, the PBMCs are
conditioned by incubating the PBMC in the presence of an
adjuvant.
Inventors: |
SHAREI; Armon R.;
(Watertown, MA) ; BERNSTEIN; Howard; (Cambridge,
MA) ; LOUGHHEAD; Scott; (Watertown, MA) ;
BOOTY; Matthew; (Cambridge, MA) ; BLAGOVIC;
Katarina; (Cambridge, MA) ; HLAVATY; Kelan;
(Belmont, MA) ; YARAR; Defne; (Watertown, MA)
; OZAY; Emrah Ilker; (Watertown, MA) ; SMITH;
Carolyne Kelly; (Waltham, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
SQZ Biotechnologies Company |
Watertown |
MA |
US |
|
|
Family ID: |
1000004958141 |
Appl. No.: |
16/803937 |
Filed: |
February 27, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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62948732 |
Dec 16, 2019 |
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62933304 |
Nov 8, 2019 |
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62886799 |
Aug 14, 2019 |
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62841089 |
Apr 30, 2019 |
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62812225 |
Feb 28, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 39/12 20130101;
A61K 2039/5154 20130101; A61K 39/39 20130101; A61K 45/06 20130101;
C12N 5/0634 20130101; A61K 2039/55561 20130101 |
International
Class: |
C12N 5/078 20060101
C12N005/078; A61K 39/39 20060101 A61K039/39; A61K 39/12 20060101
A61K039/12 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 11, 2019 |
EP |
19161964.2 |
Claims
1-3. (canceled)
4. A conditioned plurality of modified PBMCs comprising an antigen,
wherein the antigen is exogenous to the modified PBMCs.
5. (canceled)
6. The conditioned plurality of modified PBMCs of claim 4, wherein
the modified PBMCs further comprise an adjuvant.
7-8. (canceled)
9. The conditioned plurality of PBMCs of claim 4, prepared by
incubating the plurality of PBMCs with an adjuvant for a sufficient
time for the PBMCs to condition before or after introducing the
antigen to the PBMCs, thereby generating the conditioned plurality
of PBMCs comprising the antigen.
10. A plurality of modified PBMCs comprising an antigen, or an
antigen and an adjuvant, prepared by a process comprising the steps
of: a) passing a cell suspension comprising a plurality of input
PBMCs through a cell-deforming constriction, wherein a diameter of
the constriction is a function of a diameter of the input PBMCs in
the suspension, thereby causing perturbations of the input PBMCs
large enough for the antigen or the antigen and the adjuvant to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen,
or the antigen and the adjuvant for a sufficient time to allow the
antigen or the antigen and the adjuvant to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen or the antigen and the adjuvant.
11. A plurality of modified PBMCs comprising an antigen, prepared
by a process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for a nucleic acid
encoding the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the nucleic acid encoding the antigen for a
sufficient time to allow the nucleic acid encoding the antigen to
enter the perturbed input PBMCs, wherein the nucleic acid is
expressed in the PBMCs to produce the antigen thereby generating a
plurality of modified PBMCs comprising the antigen.
12. (canceled)
13. The plurality of modified PBMCs of claim 11, the process
further comprises the step of: a) incubating the plurality of input
PBMCs with an adjuvant for a sufficient time for the input PBMCs to
condition, thereby generating a conditioned plurality of input
PBMCs; and/or b) incubating the plurality of modified PBMCs
comprising the nucleic acid encoding the antigen with an adjuvant
for a sufficient time for the modified PBMCs comprising the nucleic
acid encoding the antigen to condition, wherein the nucleic acid is
expressed in the PBMCs to produce the antigen thereby generating
the conditioned plurality of modified PBMCs comprising the
antigen.
14-16. (canceled)
17. The plurality of modified PBMCs of claim 10, wherein the input
PBMCs comprise an adjuvant.
18. A plurality of modified PBMCs comprising an antigen and an
adjuvant, prepared by a process comprising the steps of: a) passing
a cell suspension comprising a plurality of input PBMCs comprising
the antigen through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the adjuvant to pass through to form a
plurality of perturbed input PBMCs; and b) incubating the plurality
of perturbed input PBMCs with the adjuvant for a sufficient time to
allow the adjuvant to enter the perturbed input PBMCs, thereby
generating the plurality of modified PBMCs comprising the antigen
and the adjuvant.
19. The plurality of modified PBMCs according to claim 10, wherein
the process further comprises: a) incubating the plurality of input
PBMCs with a second adjuvant for a sufficient time for the input
PBMCs to condition, thereby generating a conditioned plurality of
input PBMCs; and/or (b) incubating the plurality of modified PBMCs
comprising the antigen or the antigen and the adjuvant with a
second adjuvant for a sufficient time for the modified PBMCs
comprising the antigen or the antigen and the adjuvant to
condition, thereby generating a conditioned plurality of modified
PBMCs comprising the antigen or the antigen and the adjuvant.
20. The plurality of modified PBMCs of claim 10, wherein the
process further comprises a step of incubating the input PBMCs
and/or the modified PBMCs with an agent that enhances the viability
and/or function of the modified PBMCs as compared to corresponding
modified PBMCs prepared without the further incubation step.
21. The plurality of modified PBMCs of claim 10, wherein the
diameter of the constriction is about 10% to about 99% of the mean
diameter of the plurality of input PBMCs.
22. The plurality of modified PBMCs of claim 10, wherein the
diameter of the constriction is: (a) about 4.2 .mu.m to about 6
.mu.m; (b) about 4.2 .mu.m to about 4.8 .mu.m; or (c) about 4.5
.mu.m.
23. (canceled)
24. The plurality of modified PBMCs of claim 10, wherein the cell
suspension comprising the plurality of input PBMCs are is passed
through multiple constrictions wherein the multiple constrictions
are arranged in series and/or in parallel.
25. (canceled)
26. The conditioned plurality of modified PBMCs of claim 10,
wherein the plurality of modified PBMCs is incubated with the
adjuvant for about 2 hours to about 10 hours; or for about 3 hours
to about 6 hours; or for about 4 hours for the modified PBMCs to
condition.
27. The plurality of modified PBMCs of claim 10, wherein the
antigen and/or the adjuvant are present in at least about 70% of
the cells in the plurality of PBMCs.
28. The plurality of modified PBMCs of claim 10, wherein the
adjuvant is a CpG oligodeoxynucleotide (ODN), LPS, IFN-.alpha.,
STING agonists, RIG-I agonists, poly I:C, R837, R848, a TLR3
agonist, a TLR4 agonist or a TLR 9 agonist.
29. The plurality of modified PBMCs of claim 10, wherein the
adjuvant is a CpG oligodeoxynucleotide (ODN).
30. The plurality of modified PBMCs of claim 10, wherein the
antigen is a disease-associated antigen.
31. The plurality of modified PBMCs of claim 10, wherein the
antigen is a human papillomavirus (HPV) antigen.
32. The plurality of modified PBMCs of claim 10, wherein the cells
are further modified to increase expression of: (a) one or more of
co-stimulatory molecules; and/or (b) one or more cytokines.
33. The plurality of modified PBMCs of claim 32, wherein: (a) the
cells are further modified to increase expression of one or more of
co-stimulatory molecules, wherein the co-stimulatory molecule is
B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86), CD70, LIGHT, HVEM, CD40,
4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4, SLAM, CD48, CD58, CD155,
or CD112; and/or (b) the cells are further modified to increase
expression of one or more cytokines, wherein the cytokine is IL-15,
IL-12, IL-2, IFN-.alpha., or IL-21.
34-35. (canceled)
36. The plurality of modified PBMCs of claim 10, wherein: (a) one
or more co-stimulatory molecules is upregulated in the B cells of
the conditioned plurality of modified PBMCs compared to the B cells
in the plurality of unmodified PBMCs, wherein the co-stimulatory
molecule is CD80 and/or CD86; and/or (b) the modified PBMCs have
increased expression of one or more of IFN-.gamma., IL-6, MCP-1,
MIP-1.beta., IP-10, or TNF-.alpha. compared to a plurality of
unconditioned PBMCs.
37. The plurality of modified PBMCs of claim 36, wherein: (a) the
CD80 and/or CD86 is upregulated in the B cells of the conditioned
plurality of modified PBMCs by more than about 1.2-fold, 1.5-fold,
1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than
10-fold compared to the B cells in a plurality of unconditioned
PBMCs; and/or (b) the expression of one or more of IFN-.gamma.,
IL-6, MCP-1, MIP-113, IP-10, or TNF-.alpha. is increased by more
than about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold,
5-fold, 8-fold, or more than 10-fold compared to the plurality of
unconditioned PBMCs.
38-39. (canceled)
40. A composition comprising the plurality of modified PBMCs of
claim 10.
41-47. (canceled)
48. A method for stimulating an immune response in an individual,
comprising: a) incubating a plurality of PBMCs with an adjuvant for
a sufficient time for the PBMCs to condition, wherein the plurality
of PBMCs comprises an antigen before the conditioning or wherein an
antigen is introduced to the plurality of PBMCs after the
conditioning, thereby generating a conditioned plurality of PBMCs
comprising the antigen; b) administering the conditioned plurality
of PBMCs comprising the antigen to the individual.
49. (canceled)
50. A method for stimulating an immune response in an individual,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for an antigen or an antigen and an
adjuvant to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen or the antigen and the adjuvant for a sufficient time
to allow the antigen or the antigen and the adjuvant to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen or the antigen and the adjuvant; c)
administering the plurality of modified PBMCs comprising the
antigen or the antigen and the adjuvant to the individual.
51-52. (canceled)
53. The method of claim 50, wherein the PBMCs are conditioned by a
process comprising the step of: a) incubating a plurality of input
PBMCs with a second adjuvant for a sufficient time for the input
PBMCs to condition, thereby generating a conditioned plurality of
input PBMCs; and/or b) incubating the plurality of modified PBMCs
comprising the antigen or the antigen and the adjuvant with a
second adjuvant for a sufficient time for the modified PBMCs
comprising the antigen or the antigen and the adjuvant to
condition, thereby generating a conditioned plurality of modified
PBMCs comprising the antigen or the antigen and the adjuvant.
54. The method of claim 50, wherein the input PBMCs comprise an
adjuvant.
55. A method for stimulating an immune response in an individual,
comprising: a) passing a cell suspension comprising an input PBMCs
comprising an antigen through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an adjuvant to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the adjuvant for a
sufficient time to allow the adjuvant to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen and the adjuvant; and c) administering the plurality of
modified PBMCs to the individual.
56. The method of claim 50, further comprising the step of
administering an adjuvant to the individual.
57. The method of claim 55, further comprising the step of
administering an adjuvant to the individual.
58. (canceled)
59. A method for stimulating an immune response in an individual,
comprising: administering to the individual a plurality of modified
PBMCs associated with an antigen, wherein the plurality of modified
PBMCs is prepared by a process comprising the steps of: a)
incubating a plurality of input PBMCs with an antigen for a
sufficient time to allow the antigen to associate with the cell
surface of the input PBMCs, thereby generating the plurality of
modified PBMCs associated with the antigen; and b) administering
the plurality of modified PBMCs to the individual.
60. The method of claim 50, wherein the stimulation of the immune
response is for use in treating cancer, an infectious disease, or a
viral associated disease in an individual.
61. The method of claim 50, wherein the plurality of modified PBMCs
is administered prior to, concurrently with, or following
administration of: (a) a cytokine; (b) an immune checkpoint
inhibitor; and/or (c) a therapeutic agent.
62. The method of claim 61, wherein the plurality of modified PBMCs
is administered prior to, concurrently with, or following
administration of: (a) a cytokine, wherein the cytokine is IL-2;
(b) an immune checkpoint inhibitor; wherein the immune checkpoint
inhibitor is targeted to any one of PD-1, PD-L1, CTLA-4, LAG3,
VISTA, and TIM-3; and/or (c) a therapeutic agent, wherein the
therapeutic agent is a chemotherapeutic agent.
63-66. (canceled)
67. A method for generating a conditioned plurality of PBMCs
comprising an antigen, comprising incubating a plurality of PBMCs
with an adjuvant for a sufficient time for the PBMCs to condition
before or after introducing the antigen to the plurality of PBMCs,
thereby generating the conditioned plurality of PBMCs comprising
the antigen.
68-69. (canceled)
70. A method for generating a plurality of modified PBMCs
comprising an antigen, or an antigen and an adjuvant, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen or the antigen and the adjuvant to pass
through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the antigen
or the antigen and the adjuvant for a sufficient time to allow the
antigen or the antigen and the adjuvant to enter the perturbed
input PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen or the antigen and the adjuvant.
71. (canceled)
72. The method of claim 70, wherein the method further comprises:
a) incubating the plurality of input PBMCs with a second adjuvant
for a sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; and/or b)
incubating the plurality of modified PBMCs comprising the antigen
or the antigen and the adjuvant with a second adjuvant for a
sufficient time for the modified PBMCs comprising the antigen or
the antigen and the adjuvant to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen or
the antigen and the adjuvant.
73-93. (canceled)
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application claims the benefit of U.S. Provisional
Application No. 62/812,225, filed on Feb. 28, 2019, European Patent
Application No. 19161964.2, filed on Mar. 11, 2019, U.S.
Provisional Application No. 62/841,089, filed on Apr. 30, 2019,
U.S. Provisional Application No. 62/886,799, filed on Aug. 14,
2019, U.S. Provisional Application No. 62/933,304, filed on Nov. 8,
2019, and U.S. Provisional Application No. 62/948,732, filed on
Dec. 16, 2019, the entire contents of each of which are
incorporated herein by reference.
SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE
[0002] The content of the following submission on ASCII text file
is incorporated herein by reference in its entirety: a computer
readable form (CRF) of the Sequence Listing (file name:
750322002200SEQLIST.TXT, date recorded: Feb. 24, 2020, size: 15
KB).
FIELD OF THE INVENTION
[0003] The present disclosure relates generally to peripheral blood
mononuclear cells (PBMCs) comprising an antigen and/or an adjuvant,
methods of manufacturing such PBMCs cells, and methods of using
such PBMCs, such as for modulating an immune response in an
individual.
BACKGROUND OF THE INVENTION
[0004] Immunotherapy can be divided into two main types of
interventions, either passive or active. Passive protocols include
administration of pre-activated and/or engineered cells,
disease-specific therapeutic antibodies, and/or cytokines. Active
immunotherapy strategies are directed at stimulating immune system
effector functions in vivo. Several current active protocols
include vaccination strategies with disease-associated peptides,
lysates, or allogeneic whole cells, infusion of autologous DCs as
vehicles for tumor antigen delivery, and infusion of immune
checkpoint modulators. See Papaioannou, Nikos E., et al. Annals of
translational medicine 4.14 (2016).
[0005] CD8.sup.+ cytotoxic T lymphocytes (CTL) and CD4.sup.+ helper
T (Th) cells stimulated by disease-associated antigens have the
potential to target and destroy diseased cells; however, current
methods for inducing endogenous T cell responses have faced
challenges.
[0006] All references cited herein, including patent applications
and publications, are incorporated by reference in their
entirety.
BRIEF SUMMARY OF THE INVENTION
[0007] The present invention provides peripheral blood mononuclear
cells (PBMCs) comprising an antigen for the stimulation of immune
responses in individuals. In some embodiments, antigens are
delivered intracellularly using the Cell Squeeze.RTM. platform. The
present inventors have unexpectedly found that mixed populations of
PBMCs have greater efficacy than pure B cell and T cell
populations. In addition, the invention is based at least in part
on the unexpected discovery that conditioning PBMCs with an
adjuvant increased activation of antigen presenting cells of the
PBMCs leading to increased immunostimulation when the PBMCs are
administered to an individual.
[0008] In some aspects, the invention provides a plurality of
modified PBMCs comprising an antigen, wherein the antigen is
exogenous to the modified PBMCs. In some embodiments, the invention
provides a plurality of modified PMBCs comprising an antigen,
wherein the antigen is exogenous to the modified PBMCs, wherein the
antigen is a cancer antigen, an infectious disease antigen or a
viral-disease associated antigen. In some aspects, the invention
provides a conditioned plurality of modified PBMCs comprising an
antigen, wherein the antigen is exogenous to the modified PBMCs. In
some embodiments, the invention provides a conditioned plurality of
modified PMBCs comprising an antigen, wherein the antigen is
exogenous to the modified PBMCs, wherein the antigen is a cancer
antigen, an infectious disease antigen or a viral-disease
associated antigen. In some embodiments, the invention provides a
conditioned plurality of modified PBMCs comprising an antigen and
an adjuvant, wherein the antigen is exogenous to the modified
PBMCs.
[0009] In some aspects, the invention provides a conditioned
plurality of PBMCs comprising an antigen, prepared by incubating
the plurality of PBMCs comprising the antigen with an adjuvant for
a sufficient time for the PBMCs to condition, thereby generating
the conditioned plurality of PBMCs comprising the antigen. In some
embodiments, the invention provides a conditioned plurality of
PBMCs comprising an antigen, prepared by incubating the plurality
of PBMCs with an adjuvant for a sufficient time for the PBMCs to
condition prior to introducing the antigen to the PBMCs, thereby
generating the conditioned plurality of PBMCs comprising the
antigen.
[0010] In some aspects, the invention provides a plurality of
modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen. In some embodiments, the invention provides a
conditioned plurality of modified PBMCs comprising an antigen,
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with an adjuvant for a
sufficient time for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the process
further comprises: isolating the plurality of modified PBMCs
comprising the antigen from the cell suspension before incubation
with the adjuvant to condition the modified PBMCs. In some
embodiments, the invention provides a plurality of modified PBMCs
comprising an antigen and an adjuvant, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen and the adjuvant to
pass through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the antigen
and the adjuvant for a sufficient time to allow the antigen and the
adjuvant to enter the perturbed input PBMCs; thereby generating the
plurality of modified PBMCs comprising the antigen and
adjuvant.
[0011] In some aspects, the invention provides a conditioned
plurality of modified PBMCs comprising an antigen, prepared by a
process comprising the steps of: a) incubating a plurality of input
PBMCs with an adjuvant for a sufficient time for the input PBMCs to
condition, thereby generating a conditioned plurality of input
PBMCs; b) passing a cell suspension comprising the conditioned
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a conditioned plurality of perturbed input PBMCs; and c)
incubating the conditioned plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen. In some embodiments, the
invention provides a plurality of modified PBMCs comprising an
antigen and an adjuvant, prepared by a process comprising the steps
of: a) passing a cell suspension comprising a plurality of input
PBMCs comprising the adjuvant through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen and the adjuvant. In some embodiments, the
invention provides a plurality of modified PBMCs comprising an
antigen and an adjuvant, prepared by a process comprising the steps
of: a) passing a cell suspension comprising a plurality of input
PBMCs comprising the antigen through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the adjuvant to pass through to
form a plurality of perturbed input PBMCs; and b) incubating the
plurality of perturbed input PBMCs with the adjuvant for a
sufficient time to allow the adjuvant to enter the perturbed input
PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen and the adjuvant. In some embodiments, the
plurality of modified PBMCs comprising the antigen and/or the
adjuvant according as described herein, wherein the process further
comprises: incubating the plurality of modified PBMCs comprising
the antigen and/or adjuvant with a second adjuvant for a sufficient
time for the modified PBMCs comprising the antigen to condition,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen and/or the adjuvant. In some embodiments,
the process further comprises: isolating the plurality of modified
PBMCs comprising the antigen and/or the adjuvant from the cell
suspension before incubation with the adjuvant to condition the
modified PBMCs. In some embodiments, the process further comprises
a step of incubating the input PBMCs and/or the modified PBMCs with
an agent that enhances the viability and/or function of the
modified PBMCs as compared to corresponding modified PBMCs prepared
without the further incubation step.
[0012] In some aspects, the invention provides a composition
comprising the plurality of modified PBMCs as described herein for
use in a method of treatment of the human or animal body by
surgery, therapy or diagnosis. In some embodiments, the invention
provides a composition comprising the plurality of modified PBMCs
as described herein for use in the treatment of a cancer, an
infectious disease or a viral-associated disease.
[0013] In some aspects, the invention provides a composition
comprising a conditioned plurality of modified PBMCs comprising an
antigen for use as a medicament, wherein the conditioned plurality
of modified PBMCs is prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the invention provides a composition comprising a
conditioned plurality of modified PBMCs comprising an antigen for
use in a method of treatment of the human or animal body by
surgery, therapy or diagnosis, wherein the conditioned plurality of
modified PBMCs is prepared by a process comprising the steps of: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen a sufficient time to allow the antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the antigen; and c) incubating the
plurality of modified PBMCs comprising the antigen with an adjuvant
for a sufficient time for the modified PBMCs comprising the antigen
to condition, thereby generating the conditioned plurality of
modified PBMCs comprising the antigen. In some embodiments, the
invention provides a composition comprising a conditioned plurality
of modified PBMCs comprising an antigen for use as a medicament,
wherein the conditioned plurality of modified PBMCs is prepared by
a process comprising the steps of: a) incubating a plurality of
input PBMCs with an adjuvant for a sufficient time for the input
PBMCs to condition, thereby generating a conditioned plurality of
input PBMCs; b) passing a cell suspension comprising the
conditioned plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a conditioned plurality of perturbed input
PBMCs; and c) incubating the conditioned plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the invention provides composition comprising a
conditioned plurality of modified PBMCs comprising an antigen for
use in a method of treatment of the human or animal body, wherein
the conditioned plurality of modified PBMCs is prepared by a
process comprising the steps of: a) incubating a plurality of input
PBMCs with an adjuvant for a sufficient time for the input PBMCs to
condition, thereby generating a conditioned plurality of input
PBMCs; b) passing a cell suspension comprising the conditioned
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a conditioned plurality of perturbed input PBMCs; and c)
incubating the conditioned plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen. In some embodiments, the
invention provides a composition comprising a conditioned plurality
of modified PBMCs comprising an antigen for use in a method of
treating cancer an infectious disease or a viral associated disease
in an individual, wherein the conditioned plurality of modified
PBMCs is prepared by a process comprising the steps of: a) passing
a cell suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with an adjuvant for a
sufficient time for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the invention
provides a composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in the treatment of
cancer, an infectious disease or a viral associated disease in an
individual, wherein the conditioned plurality of modified PBMCs is
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a cell
deforming constriction, wherein a diameter of the constriction is a
function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with an adjuvant for a
sufficient time for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the invention
provides a composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treating a HPV-associated disease in an individual, wherein the
conditioned plurality of modified PBMCs is prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with an adjuvant for a sufficient time for
the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the invention provides a
composition comprising a conditioned plurality of modified PBMCs
comprising an antigen for use in the treatment of a HPV-associated
disease in an individual, wherein the conditioned plurality of
modified PBMCs is prepared by a process comprising the steps of: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0014] In some aspects, the invention provides the use of a
composition comprising a conditioned plurality of modified PBMCs
comprising an antigen in the manufacture of a medicament for
treating cancer, an infectious disease or a viral-associated
disease in an individual, wherein the conditioned plurality of
modified PBMCs is prepared by a process comprising the steps of: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the invention provides the use of a composition
comprising a conditioned plurality of modified PBMCs comprising an
antigen in the manufacture of a medicament for treating a
HPV-associated disease, wherein the conditioned plurality of
modified PBMCs is prepared by a process comprising the steps of: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0015] In some aspects, the invention provides a conditioned
plurality of modified PBMCs comprising an antigen, prepared by a
process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is about 3
.mu.m to about 10 .mu.m, thereby causing perturbations of the input
PBMCs large enough for the antigen to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the antigen for a sufficient time to
allow the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen;
and c) incubating the plurality of modified PBMCs comprising the
antigen with an adjuvant for a sufficient time for the modified
PBMCs comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the invention provides a conditioned plurality of
modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, wherein the adjuvant is CpG
ODN, thereby generating the conditioned plurality of modified PBMCs
comprising the antigen. In some embodiments, the invention provides
a conditioned plurality of modified PBMCs comprising an antigen,
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with an adjuvant for about 1 hour to about
24 hours for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the invention
provides a conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of: a) passing
a cell suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with an adjuvant for about 1 hour to about
24 hours for the modified PBMCs comprising the antigen to
condition, wherein the adjuvant is CpG ODN, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
a preferred embodiment, the adjuvant is CPG 7909.
[0016] In some aspects, the invention provides a conditioned
plurality of modified PBMCs comprising a human papillomavirus (HPV)
antigen, prepared by a process comprising the steps of: a) passing
a cell suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the HPV antigen to pass through
to form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the HPV antigen for a
sufficient time to allow the HPV antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the HPV antigen; and c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, thereby generating the conditioned plurality of
modified PBMCs comprising the HPV antigen. In some embodiments, the
invention provides a conditioned plurality of modified PBMCs
comprising a HPV antigen, prepared by a process comprising the
steps of: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; and c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for a sufficient time for the modified PBMCs comprising the HPV
antigen to condition, wherein the CpG ODN is CpG 7909, thereby
generating the conditioned plurality of modified PBMCs comprising
the HPV antigen. In some embodiments, the invention provides a
conditioned plurality of modified PBMCs comprising a HPV antigen,
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the HPV antigen to pass through
to form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the HPV antigen for a
sufficient time to allow the HPV antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the HPV antigen; and c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for about
1 hour to about 24 hours for the modified PBMCs comprising the HPV
antigen to condition, thereby generating the conditioned plurality
of modified PBMCs comprising the HPV antigen. In some embodiments,
the invention provides a conditioned plurality of modified PBMCs
comprising a HPV antigen, prepared by a process comprising the
steps of: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; and c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for about 1 hour to about 24 hours for the modified PBMCs
comprising the HPV antigen to condition, wherein the CpG ODN is CpG
7909, thereby generating the conditioned plurality of modified
PBMCs comprising the HPV antigen.
[0017] In some aspects, the invention provides a conditioned
plurality of modified PBMCs comprising an antigen, prepared by a
process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is about 3
.mu.m to about 10 .mu.m, thereby causing perturbations of the input
PBMCs large enough for the antigen to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the antigen for a sufficient time to
allow the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen;
and c) incubating the plurality of modified PBMCs comprising the
antigen with a CpG ODN for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the invention provides a conditioned plurality of
modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 34 .mu.m to about
10 .mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with a CpG ODN for a sufficient time for the modified PBMCs
comprising the antigen to condition, wherein the CpG ODN is CpG
7909, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the invention
provides a conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of: a) passing
a cell suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 34 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for about 1 hour to about 24
hours for the modified PBMCs comprising the antigen to condition,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen. In some embodiments, the invention provides
a conditioned plurality of modified PBMCs comprising an antigen,
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for about 1 hour to about 24
hours for the modified PBMCs comprising the antigen to condition,
wherein the CpG ODN is CpG 7909, thereby generating the conditioned
plurality of modified PBMCs comprising the antigen.
[0018] In some aspects, the invention provides a conditioned
plurality of modified PBMCs comprising an antigen, prepared by a
process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is about 3
.mu.m to about 10 .mu.m, thereby causing perturbations of the input
PBMCs large enough for the antigen to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the antigen for a sufficient time to
allow the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen;
and c) incubating the plurality of modified PBMCs comprising the
antigen with a CpG ODN for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the invention provides a conditioned plurality of
modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with a CpG ODN for a sufficient time for the modified PBMCs
comprising the antigen to condition, wherein the CpG ODN is CpG
7909, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the invention
provides a conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of: a) passing
a cell suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for about 1 hour to about 24
hours for the modified PBMCs comprising the antigen to condition,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen. In some embodiments, the invention provides
a conditioned plurality of modified PBMCs comprising an antigen,
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for about 1 hour to about 24
hours for the modified PBMCs comprising the antigen to condition,
wherein the CpG ODN is CpG 7909, thereby generating the conditioned
plurality of modified PBMCs comprising the antigen.
[0019] In some aspects, the invention provides a method for
stimulating an immune response in an individual, comprising: a)
administering a plurality of modified PBMCs comprising an antigen
comprising the amino acid sequence of any one of SEQ ID NOs: 18-25
to the individual; and b) administering an adjuvant to the
individual.
[0020] In some aspects, the invention provides a method for
stimulating an immune response in an individual, comprising: a)
administering a plurality of modified PBMCs comprising an antigen
comprising the amino acid sequence of SEQ ID NO: 19 to the
individual; and b) administering an adjuvant to the individual. In
some aspects, the invention provides a method for stimulating an
immune response in an individual, comprising: a) administering a
plurality of modified PBMCs comprising an antigen comprising the
amino acid sequence of SEQ ID NO: 23 to the individual; and b)
administering an adjuvant to the individual. In some aspects, the
invention provides a method for stimulating an immune response in
an individual, comprising: a) administering a plurality of modified
PBMCs comprising a plurality of antigens comprising the amino acid
sequences of SEQ ID NO: 19 and/or SEQ ID NO:23 to the individual;
and b) administering an adjuvant to the individual. In some
aspects, the invention provides a method for stimulating an immune
response in an individual, comprising: a) administering a plurality
of modified PBMCs comprising a plurality of antigens consisting of
the amino acid sequences of SEQ ID NO: 19 and SEQ ID NO:23 to the
individual; and b) administering an adjuvant to the individual. In
some embodiments, the plurality of antigens is contained within a
pool of non-covalently linked peptides. In some embodiments, the
plurality of antigens is contained within a pool of non-covalently
linked peptides, wherein each peptide comprises no more than one
antigen. In some embodiments, the plurality of antigens is
contained within a pool of non-covalently linked peptides, wherein
the amino acid sequence of SEQ ID NO: 19 and the amino acid
sequence of SEQ ID NO: 23 are contained within separate
peptides.
[0021] In some aspects, the invention provides a method for
stimulating an immune response in an individual, comprising: a)
incubating a plurality of PBMCs comprising an antigen with an
adjuvant for a sufficient time for the PBMCs to condition, thereby
generating a conditioned plurality of PBMCs comprising the antigen;
b) administering the conditioned plurality of PBMCs comprising the
antigen to the individual. In some embodiments, the invention
provides a method for stimulating an immune response in an
individual, comprising: a) incubating a plurality of PBMCs with an
adjuvant for a sufficient time for the PBMCs to condition, thereby
generating a conditioned plurality of PBMCs comprising the antigen;
b) introducing an antigen to the plurality of PBMCs; and c)
administering the conditioned plurality of PBMCs comprising the
antigen to the individual. In some embodiments, the invention
provides a method for stimulating an immune response in an
individual, comprising: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; c) incubating the plurality of modified PBMCs
comprising the antigen with an adjuvant for a sufficient time for
the modified PBMCs comprising the antigen to condition, thereby
generating a conditioned plurality of modified PBMCs comprising the
antigen; and d) administering the conditioned plurality of modified
PBMCs comprising the antigen to the individual. In some
embodiments, the method further comprises isolating the plurality
of modified PBMCs comprising the antigen from the cell suspension
before incubation with the adjuvant. In some embodiments, the
invention provides a method for stimulating an immune response in
an individual, comprising: a) passing a cell suspension comprising
a plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an antigen and an adjuvant to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
and the adjuvant for a sufficient time to allow the antigen and the
adjuvant to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen and adjuvant;
and c) administering the plurality of modified PBMCs to the
individual.
[0022] In some aspects, the invention provides a method for
stimulating an immune response in an individual, comprising: a)
incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; c) incubating the conditioned
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen; and d) administering the conditioned
plurality of modified PBMCs to the individual. In some embodiments,
the invention provides a method for stimulating an immune response
in an individual, comprising: a) passing a cell suspension
comprising a plurality of input PBMCs comprising an adjuvant
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen and the
adjuvant; and c) administering the plurality of modified PBMCs to
the individual. In some embodiments, the invention provides a
method for stimulating an immune response in an individual,
comprising: a) passing a cell suspension comprising an input PBMCs
comprising an antigen through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an adjuvant to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the adjuvant for a
sufficient time to allow the adjuvant to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen and the adjuvant; and c) administering the plurality of
modified PBMCs to the individual. In some embodiments, the
invention provides a method for stimulating an immune response in
an individual, comprising: a) passing a cell suspension comprising
a plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; c) administering the plurality of modified PBMCs to
the individual; and d) administering an adjuvant to the individual.
In some embodiments, the invention provides a method for
stimulating an immune response in an individual, comprising: a)
passing a cell suspension comprising an input PBMCs comprising an
antigen through a cell-deforming constriction, wherein a diameter
of the constriction is a function of a diameter of the input PBMCs
in the suspension, thereby causing perturbations of the input PBMCs
large enough for an adjuvant to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the adjuvant for a sufficient time to allow the
adjuvant to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen and the
adjuvant; and c) administering the plurality of modified PBMCs to
the individual; and d) administering an adjuvant to the
individual.
[0023] In some aspects, the invention provides a method for
stimulating an immune response in an individual, comprising:
administering to the individual a plurality of PBMCs associated
with an antigen, wherein the plurality of modified PBMCs is
prepared by a process comprising the steps of: a) incubating a
plurality of input PBMCs with an antigen for a sufficient time to
allow the antigen to associate with the cell surface of the input
PBMCs, thereby generating the plurality of PBMCs associated with
the antigen; and b) administering the plurality of modified PBMCs
to the individual. In some embodiments, the method further
comprises administering an adjuvant to the individual.
[0024] In some aspects, the invention provides a method for
generating a conditioned plurality of PBMCs comprising an antigen,
comprising incubating a plurality of PBMCs comprising the antigen
with an adjuvant for a sufficient time for the PBMCs to condition,
thereby generating the conditioned plurality of PBMCs comprising
the antigen. In some embodiments, the invention provides a method
for generating a conditioned plurality of modified PBMCs comprising
an antigen, comprising: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with an adjuvant for a sufficient time for
the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the method further comprises
isolating the plurality of modified PBMCs comprising the antigen
from the cell suspension before incubation with the adjuvant. In
some embodiments, the invention provides a method for generating a
plurality of modified PBMCs comprising an antigen, comprising: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; and b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen. In some
embodiments, the invention provides a method for generating a
plurality of modified PBMCs comprising an antigen and an adjuvant,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the antigen and the adjuvant to pass
through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the antigen
and the adjuvant for a sufficient time to allow the antigen and the
adjuvant to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen and adjuvant. In
some embodiments, the invention provides a method of generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) incubating a plurality of input PBMCs with an
adjuvant for a sufficient time for the input PBMCs to condition,
thereby generating a conditioned plurality of input PBMCs; b)
passing a cell suspension comprising the conditioned plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the antigen to pass through to form a
conditioned plurality of perturbed input PBMCs; and c) incubating
the conditioned plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating the conditioned plurality of
modified PBMCs comprising the antigen. In some embodiments, the
invention provides a method for generating a plurality of modified
PBMCs comprising an antigen and an adjuvant, comprising: a) passing
a cell suspension comprising a plurality of input PBMCs comprising
an adjuvant through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for an antigen to pass through to form a
plurality of perturbed input PBMCs; and b) incubating the plurality
of perturbed input PBMCs with the antigen for a sufficient time to
allow the antigen to enter the perturbed input PBMCs, thereby
generating the plurality of modified PBMCs comprising the antigen
and the adjuvant. In some embodiments, the invention provides a
method for generating a plurality of modified PBMCs comprising an
antigen and an adjuvant, comprising: a) passing a cell suspension
comprising a plurality of input PBMCs comprising an antigen through
a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an adjuvant to pass through to form a plurality of
perturbed input PBMCs; and b) incubating the plurality of perturbed
input PBMCs with the adjuvant for a sufficient time to allow the
adjuvant to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen and the
adjuvant. In some embodiments, the method further comprises a step
of incubating the input PBMCs and/or the modified PBMCs with an
agent that enhances the viability and/or function of the modified
PBMCs as compared to corresponding modified PBMCs prepared without
the further incubation step.
BRIEF DESCRIPTION OF THE DRAWINGS
[0025] FIG. 1A is a schematic diagram of a representative cell
within a plurality of PBMCs, displaying the SQZ-mediated delivery
of E6 and/or E7 SLP; and the subsequent processing and presentation
of E6 and E7 epitopes, respectively, on MHC-I. The representative
cell can be any one of the PBMC cell types (such as T cells,
monocytes, NK cells, and B cells).
[0026] FIG. 1B is a schematic representative of cohorts in the
escalation phase in the monotherapy administration of PBMCs
comprising an HPV antigen to an individual, with or without
co-administration of a CpG adjuvant. The amount of circles depicts
relative doses of modified PBMCs, arrows depict administrations,
double helix represents CpG adjuvant, and "AVB" indicates
additional vaccine boosts.
[0027] FIG. 1C is a schematic representative of cohorts in the
escalation phase in the combination administration of PBMCs
comprising an HPV antigen and atezolizumab to an individual. The
amount of circles depicts relative doses of modified PBMCs, arrows
depict administrations, and "AVB" indicates additional vaccine
boosts.
[0028] FIG. 2A shows the viability of subpopulations in splenocytes
after incubation of dextran (endocytosis) and SQZ-mediated delivery
of dextran under a driving pressure of 30, 60, and 90 psi. FIG. 2B
shows the percentage of cells with dextran delivered by endocytosis
or SQZ-mediated delivery under a driving pressure of 30, 60, and 90
psi.
[0029] FIG. 3 shows the effects of cell conditioning and of
co-administration of CpG on the antigen-specific immune response
elicited by B cells or splenocytes SQZ-loaded with OVA.
[0030] FIG. 4 shows the effects of splenocyte conditioning and of
co-administration of CpG at various concentrations on the
antigen-specific immune response elicited by crafted splenocytes
SQZ-loaded with OVA.
[0031] FIG. 5 shows the effects of splenocyte conditioning and of
co-administration of CpG or IFN.alpha. on the antigen-specific
immune response elicited by crafted splenocytes SQZ-loaded with
OVA.
[0032] FIG. 6 shows the antigen-specific immune response elicited
by conditioned crafted splenocytes SQZ-loaded with OVA when
administered at different cell doses, with one administration
(Prime) or two administrations (Prime-boost).
[0033] FIG. 7 shows the antigen-specific immune response elicited
by (a) conditioned B cells SQZ-loaded with OVA or (b) conditioned
crafted splenocytes SQZ-loaded with OVA when administered at
different doses, with one administration (Prime) or two
administrations (Prime-boost).
[0034] FIG. 8 shows the effect of duration of conditioning (CpG
incubation) on antigen-specific immune response elicited by matured
splenocytes SQZ-loaded with OVA.
[0035] FIG. 9 shows the dose-dependent effect of matured
splenocytes SQZ-loaded with E7 HPV antigen in inhibiting
E7-expressing TC1 tumors.
[0036] FIG. 10A shows the effect of CpG incubation on activation
markers in B cell subpopulation within human PBMCs (top) and murine
splenocytes (bottom).
[0037] FIG. 10B shows the changes in cytokine/chemokine profiles
when human PBMCs or murine splenocytes were subjected to incubation
with CpG.
[0038] FIG. 11 shows the changes in cell composition and MHC-I
levels when human PBMCs were subjected to SQZ-mediated delivery at
a driving pressure of 60 psi and a constriction width of 3.5 .mu.m
or 4 .mu.m.
[0039] FIG. 12A shows the viability and payload delivery to
CD3.sup.+ T cells or CD14.sup.+ monocytes within human PBMCs that
were subjected to SQZ-mediated delivery at a driving pressure of 60
psi and with a constriction width of 3.5 .mu.m or 4 .mu.m.
[0040] FIG. 12B shows the viability and payload delivery to
CD20.sup.+ B cells or CD56.sup.+ NK cells within human PBMCs that
were subjected to SQZ-mediated delivery at a driving pressure of 60
psi and with a constriction width of 3.5 .mu.m or 4 .mu.m.
[0041] FIG. 13 shows the correlation of delivery in subsets with
PBMC population (bottom) with the stimulation of E7-specific
responder cells (top) when co-cultured with matured human PBMCs
loaded with an E7 HPV antigen via a SQZ-mediated process using a
3.5 .mu.m or 4 .mu.m constriction width.
[0042] FIG. 14 shows the stimulation of E7-specific responder cells
when co-cultured with matured human PBMCs loaded with an E7 HPV
antigen via a SQZ-mediated process with a driving pressure of 45
psi or 60 psi, with constriction widths of 3.5 .mu.m, 4 .mu.m or
4.5 .mu.m, and with the process carried out at RT (top) or on ice
(bottom).
[0043] FIG. 15 top panel shows the stimulation of pp65-specific
responder cells, when co-cultured (in the presence or absence of 1
.mu.M CpG) with human PBMCs SQZ-loaded with pp65 or human PBMCs
SQZ-loaded with pp65 and matured with adjuvant.
[0044] FIG. 15 bottom panel shows the stimulation of pp65-specific
responder cells (a) when co-cultured, in the presence of 1 .mu.M
CpG, with human PBMCs SQZ-loaded with pp65 or human PBMCs
SQZ-loaded with pp65 and matured with adjuvant; or (b) when
co-cultured with human T cell SQZ-loaded with pp65 or human T cells
SQZ-loaded with pp65 and matured with adjuvant.
[0045] FIG. 16 shows the effects of (a) PBMC maturation by various
adjuvants and (b) pp65 antigen concentration used in SQZ loading on
the stimulation of pp65-specific responder cells, when responders
were co-cultured with matured human PBMCs SQZ-loaded with pp65 CMV
antigen.
[0046] FIG. 17 shows the effects of PBMC maturation by different
adjuvants CpG, R837, and R848 with incubation times of 3 or 24
hours, on the stimulation of pp65-specific responder cells when
co-cultured with matured human PBMCs SQZ-loaded with pp65 CMV
antigen.
[0047] FIG. 18 shows the effect of conditioning of splenocytes,
before versus after SQZ-mediated loading, on the antigen-specific
response elicited by conditioned murine splenocytes SQZ-loaded with
pp65 CMV antigen.
[0048] FIG. 19 shows the effect on tumor inhibition when mice
carrying E7-expressing tumor was administered with pp65-loaded
splenocytes alone (group B); chemotherapy (cisplatin) alone (groups
C, D, G); or pp65-loaded splenocytes in combination with
chemotherapy (groups E, F, H).
[0049] FIG. 20A shows the efficiency of SQZ-mediated delivery of 3
kDa dextran to subpopulations of human PBMCs at room temperature or
on ice. FIG. 20B shows the efficiency of SQZ-mediated delivery of 3
kDa dextran to subpopulations of human PBMCs at a driving pressure
of 50 psi or 70 psi.
[0050] FIGS. 21A and 21B shows the effect of splenocyte
conditioning or CpG co-administration on tumor inhibition (FIG.
21A) and survival improvement (FIG. 21B) when mice carrying an
E7-expressing tumor was administered with crafted murine
splenocytes SQZ-loaded with E7 HPV antigen.
[0051] FIG. 22 shows the effect of splenocyte conditioning, with or
without co-administration with CpG or IFN.alpha., on the
antigen-specific response elicited by crafted murine splenocytes
SQZ-loaded with OVA antigen.
[0052] FIGS. 23A-23H show the effect of splenocyte conditioning,
with or without incubation with CpG 1826, on the expression of B
cell markers CD86 (FIGS. 23A-D) and H-2Kb (FIGS. 23E-H) within
crafted murine splenocytes that were either subjected to
SQZ-processing or unprocessed.
[0053] FIGS. 24A-24D show the levels of circulating cytokines in
untreated mice (FIG. 24A), mice injected with 1 .mu.g CpG 1826 IV
only (FIG. 24B), mice immunized with crafted murine splenocytes
SQZ-loaded with E7 SLP (FIG. 24C), or mice co-injected with crafted
murine splenocytes SQZ-loaded with E7 SLP and co-injected with 1
.mu.g CpG 1826 IV (FIG. 24D).
[0054] FIG. 25 shows the circulation kinetics of crafted murine
splenocytes SQZ-loaded with E7 SLP (M-SQZ-Spleno-HPV) and
unprocessed crafted murine splenocytes.
[0055] FIGS. 26A-26E show the amount of E7-specific T cell
infiltration and FIG. 26F shows the tumor volume over time after
immunization with crafted murine splenocytes SQZ-loaded with E7 SLP
or unprocessed crafted murine splenocytes. The percentage of CD8+ T
cells per live cells in tumor environment, and the number of CD8+ T
cells per tumor mass are shown in FIGS. 26A and 26D respectively.
The percentage of E7-specific T cells per live cells in tumor
environment, and the number of E7-specific T cells per tumor mass
are shown in FIGS. 26C and 26E respectively. The percentage of
E7-specific T cells per CD8+ T cells in the tumor environment is
shown in FIG. 26B.
[0056] FIGS. 27A-27D show the amount of in vivo proliferation of
OVA-specific T cell (OT-I CD8.sup.+ T cell) in WT or MHC-I -/-
mice, with stimulation by crafted murine splenocytes SQZ-loaded
with OVA, or by crafted murine splenocytes incubated with OVA
(Incubation Control) or no stimulation (OT-I Control). FIGS. 27A
and 27C show the respective OT-I proliferation in recipient lymph
nodes for 2 replicate experiments. FIGS. 27B and 27D show the
respective OT-I proliferation in recipient spleens for 2 replicate
experiments.
[0057] FIGS. 28A-28E show the proliferation and expression of
activation marker CD69 in OT-I CD8.sup.+ T cell after co-culturing
with crafted murine splenocytes SQZ-loaded with OVA (FIG. 28A), or
with the indicated subsets of crafted murine splenocytes SQZ-loaded
with OVA (subsets of B cells, T cells, monocytes, NK cells for
FIGS. 28B, 28C, 28D, 28E respectively).
[0058] The top panels in FIGS. 29A-29F show confocal imaging from
middle of a Z-stack for each sample, demonstrating localization of
plasma membrane (CD45 staining, PM, top panels); the localization
of FAM-labeled HPV SLPs (SLP, second panels from top); and the
overlay showing their relative localization (Overlay, third panels
from top), whereas the bottom panels in FIG. 29 show line traces
across the center of the cell along the white lines shown in the
respective overlay panels, for human PBMCs SQZ-processed in RPMI
(FIG. 29A, 29C, 29E) or human PBMCs SQZ-loaded with FAM-labeled E6,
E7, or E6+E7 SLPs (FIGS. 29B, 29D, 29F respectively).
[0059] FIG. 30 shows the proliferation of gp100 specific T cells
after co-injection of B cells either left untreated (NC), incubated
with gp100 SLP (Incub. ctrl), SQZ-loaded with gp100 SLP (Squeeze),
or pulsed with gp100 SLP (PP), as measured by CFSE dilution (left
panel) and subsequent quantification (right panel).
[0060] FIG. 31 shows the tumor volume change of implanted TC-1
tumors in mice that were untreated or prophylactically administered
with crafted splencoytes SQZ-loaded with E7 SLP. Untreated cohort I
was implanted with TC-1 tumors on Day 0. Untreated cohort II was
implanted with TC-1 tumors on Day 60. Mice treated with SQZ-loaded
splenocytes were implanted with TC-1 tumors on both Day 0 and Day
60.
[0061] FIG. 32 shows the tumor volume changes of implanted TC-1
tumors following therapeutic treatment of crafted splenocytes
SQZ-loaded with E7 SLP. Mice were treated with 0.1.times.10.sup.6
or 1.0.times.10.sup.6 SQZ-loaded splenocytes, administered either
as a single dose of priming (on Day 10 post-implantation), or under
a prime and boost regimen (Prime on Day 10, boost on Days 17 and 24
post-implantation).
[0062] FIGS. 33A and B shows the effect of splenocyte conditioning
or CpG co-administration on tumor inhibition (FIG. 33A) and
survival improvement (FIG. 33B) when mice carrying an E7-expressing
tumor was administered with crafted murine splenocytes SQZ-loaded
with HPV E7 antigen.
[0063] FIG. 34 shows the extent of MHC-I presentation of epitope
SIINFEKL(SEQ ID NO: 54) processed from OVA in splenocyte
subpopulations of T cells, B cells, NK cells and monocytes after
crafted splenocytes were SQZ-processed without cargo (SQZ only) or
SQZ-processed in the presence of OVA (SQZ+OVA).
[0064] FIG. 35 shows the dose-dependent efficacy of splenocyte
administration, for the antigen-specific response elicited by
crafted murine splenocytes SQZ-loaded with HPV16 E7 antigen.
[0065] FIGS. 36A-36C show the amount of E7-specific T cell
infiltration and FIG. 36D shows the tumor volume over time after
immunization with crafted murine splenocytes SQZ-loaded with E7
SLP, or with a peptide vaccine, or left untreated. The percentage
of CD45+ leukocytes per live cells in tumor environment, the number
of CD8+ T cells out of the CD45+ cells, and the in percentage of
E7-specific T cells per CD8+ T cells in tumor environment are shown
in FIGS. 36A, 36B and 36C respectively.
[0066] FIG. 37A shows the number of cells SQZ-processed in a
typical research setting versus the number of cells SQZ-processed
in a manufacturing setting. FIG. 37B shows the viability of PBMCs
after incubation with Dextran, or after SQZ-processing in the
presence of Dextran. FIG. 37C shows the percentage of cells
positive with Dextran after incubation, or SQZ-processing with
Dextran, for PBMCs as well as the component cell types of B cells
(CD20+), T cells (CD3+), NK cells (CD56+) and monocytes
(CD14+).
[0067] FIG. 38A shows the percentage of CD86-expressing cells
within PBMC subpopulations of B cells (CD19+), T cells (CD3+), NK
cells (CD56+) and monocytes (CD14+), after PBMCs were either left
untreated (NC), SQZ-processed with empty payload (Empty SQZ), or
SQZ-loaded with CD86-encoding mRNA (SQZ). FIG. 38B shows the
percentage of IFN.alpha.2-expressing cells within PBMC
subpopulations of B cells (CD19+), T cells (CD3+), NK cells (CD56+)
and monocytes (CD14+), after PBMCs were either SQZ-processed with
empty payload (Empty SQZ), or SQZ-loaded with IFN.alpha.2-encoding
mRNA (SQZ).
[0068] FIG. 39A shows the percentage of CD86-expressing within PBMC
subpopulation of T cells (CD3+) over 72 hours, after PBMCs were
either SQZ-processed with empty payload (Empty SQZ), or SQZ-loaded
with CD86-encoding mRNA (SQZ. FIG. 39B shows the percentage of
4-1BBL-expressing cells within PBMC subpopulation of T cells (CD3+)
over 72 hours, after PBMCs were either SQZ-processed with empty
payload (Empty SQZ), or SQZ-loaded with 4-1BBL-encoding mRNA
(SQZ).
[0069] FIG. 40 shows the amount of eGFP expression within PBMC
subpopulation of T cells (CD3+) after PBMCs were either
SQZ-processed with an unmodified eGFP mRNA or with an eGFP mRNA
carrying a 5-metoxyuridine backbone modification (5moU), at mRNA
concentrations of 0 .mu.g/mL to 200 .mu.g/mL.
[0070] FIG. 41 shows the degree of secretion of IL-12, IFN.alpha.
or IL-2 cytokine by PBMCs, after PBMCs were either left untreated
(NC) or SQZ-processed with mRNA encoding IL-12, IFN.alpha. or IL-2
respectively (SQZ).
[0071] FIG. 42A shows the schematics of Signals 1, 2, 3 from an
enhanced antigen presenting cell in stimulating an effector immune
cell response. FIG. 42B shows the amount of Signal 2 effector
expression over 48 hours within PBMC subpopulation of B cells
(CD19+), T cells (CD3+), NK cells (CD56+) and monocytes (CD14+),
after PBMCs were SQZ-processed with mRNA encoding CD70 or 4-1BBL
respectively. FIG. 42C shows the amount of Signal 3 effector
secretion by PBMCs over 24 hours, after PBMCs were SQZ-processed
with mRNA encoding IFN.alpha.2 or IL-2 respectively.
[0072] FIG. 43A shows the amount of eGFP translation and expression
in PBMCs, for PBMCs that were unstimulated, stimulated with ConA
before SQZ-processing, stimulated with conA after SQZ-processing,
where the PBMCs were SQZ-processed in the presence of eGFP-encoding
mRNA. FIG. 43B shows the amount of CD86 expression in PBMCs that
were unstimulated, or stimulated with ConA before SQZ-processing,
when the PBMCs were SQZ-processed in the presence of CD86-encoding
mRNA
[0073] FIG. 44A shows the schematics of an experiment in studying
whether Signal 2 and Signal 3 Effector mRNAs were translated in
crafted mouse splenocytes subsequent to SQZ-loading. FIG. 44B shows
the amount of CD70, CD80, CD86 or OX40L expression in crafted
murine splenocytes after the crafted splenocytes were SQZ-processed
in the presence of mRNAs encoding CD70, CD80, CD86 or OX40L
respectively. FIG. 44C shows the amount of IL-12, IL-2 or
IFN.alpha.2 secretion by crafted murine splenocytes after the
crafted splenocytes were SQZ-processed in the presence of mRNAs
encoding IL-12, IL-2 or IFN.alpha.2 respectively.
[0074] FIG. 45A shows the schematics of an experiment in studying
whether SQZ-loading of Signal 2 and Signal 3 Effector mRNAs in
crafted murine splenocytes could facilitate an enhanced ability to
stimulate antigen-specific T cell response. FIG. 45B showed the
degree of activation of OVA-specific T cells upon co-culture with
crafted murine splenocytes SQZ-loaded with OVA peptide and mRNA
encoding Signal 2 effectors (CD70, CD80 or CD86). FIG. 45C showed
the degree of activation of OVA-specific T cells upon co-culture
with crafted murine splenocytes SQZ-loaded with OVA peptide and
mRNA encoding Signal 3 effector IL-2.
[0075] FIG. 46A shows the amount of activation of antigen-specific
responder T cells upon co-culture with human PBMCs SQZ-loaded with
mRNAs encoding the respective antigens (E7, HSV GD1, MART-1, pp65
or Influenza M1). FIGS. 46B and 46C shows the amount of translation
and expression of E7 or M1 in PBMCs, after PBMCs were SQZ-processed
in the presence of mRNAs encoding E7 or M1 respectively.
[0076] FIG. 47A shows the schematics of an experiment in comparing
SQZ-loading of mRNA versus protein form of antigen with regards to
potency of loaded murine splenocytes in stimulating an
antigen-specific T cell response. FIG. 47B shows the amount of
activated OVA-specific T cells upon co-culture with murine
splenocytes SQZ-loaded with OVA protein or OVA-encoding mRNA.
[0077] FIGS. 48A and 48B show the experimental design and
schematics, respectively, of an experiment in studying whether
combination of of E7-loaded crafted splenocytes and anti-CTLA4
administration will result in improved therapeutic effect against
E7-carrying tumor TC1. FIGS. 48C, 48D, 48E and 48F show the
additive therapeutic effect of combination of tumor antigen-loaded
splenocytes and immune checkpoint inhibitor on tumor growth
inhibition (FIG. 48C), delay or inhibition on tumor occurrence
(FIGS. 48D and 48E) and survival improvement (FIG. 48F) when mice
carrying an E7-expressing tumor was administered with crafted
murine splenocytes SQZ-loaded with HPV E7 antigen with or without
further administration of anti-CTLA4 antibody.
DETAILED DESCRIPTION OF THE INVENTION
[0078] Antigen presenting cells (APCs) play a key role in inducing
endogenous activation of CTLs. In this work, the implementation of
the Cell Squeeze.RTM. platform to engineer peripheral blood
mononuclear cells (PBMCs) for use in modulating an immune response
to various indications, including cancer and infectious disease, is
described. By enabling efficient cytosolic delivery of target
antigens and/or adjuvants to PBMCs, this platform has demonstrated
the ability to induce highly effective MHC-I presentation of target
antigens and stimulation of CTLs in vivo. The present inventors
have unexpectedly discovered that mixed populations of PBMCs have
greater efficacy that pure B cell and T cell populations alone. In
addition, the inventors unexpectedly discovery that conditioning
PBMCs with an adjuvant increased the activation of antigen
presenting cells leading to greater immunostimulation when
administered to an individual compared to non-conditioned
antigen-loaded PBMCs.
[0079] The present application, in some aspects, provides modified
PBMCs comprising an antigen and an adjuvant, and wherein the
antigen is present intracellularly. In some embodiments, the PBMCs
comprising the antigen are incubated in the presence of an adjuvant
for a period of time prior to administration to an individual
(i.e., the PBMCs are conditioned). In some embodiments, the PBMCs
are incubated in the presence of an adjuvant for a period of time
prior to introducing the antigen to the PBMCs.
[0080] In some embodiments, the modified PBMCs are prepared by a)
passing an input PBMC cell suspension through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs, thereby causing perturbations of
the input PBMCs cell large enough for the antigen to pass through
to form a perturbed input PBMCs; and b) incubating the perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs; thereby generating the
modified PBMCs comprising the antigen. Also provided are methods of
using the modified PBMCs for modulating an immune response in an
individual, for example, for enhancing an immune response in the
individual. In some embodiments, the enhanced immune response is
directed towards the antigen. In some embodiments, the
cell-deforming constriction is contained in a microfluidic channel,
such as any of the microfluidic channels described herein.
General Techniques
[0081] The techniques and procedures described or referenced herein
are generally well understood and commonly employed using
conventional methodology by those skilled in the art, such as, for
example, the widely utilized methodologies described in Molecular
Cloning: A Laboratory Manual (Sambrook et al., 4th ed., Cold Spring
Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2012); Current
Protocols in Molecular Biology (F. M. Ausubel, et al. eds., 2003);
the series Methods in Enzymology (Academic Press, Inc.); PCR 2: A
Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor
eds., 1995); Antibodies, A Laboratory Manual (Harlow and Lane,
eds., 1988); Culture of Animal Cells: A Manual of Basic Technique
and Specialized Applications (R. I. Freshney, 6th ed., J. Wiley and
Sons, 2010); Oligonucleotide Synthesis (M. J. Gait, ed., 1984);
Methods in Molecular Biology, Humana Press; Cell Biology: A
Laboratory Notebook (J. E. Cellis, ed., Academic Press, 1998);
Introduction to Cell and Tissue Culture (J. P. Mather and P. E.
Roberts, Plenum Press, 1998); Cell and Tissue Culture: Laboratory
Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J.
Wiley and Sons, 1993-8); Handbook of Experimental Immunology (D. M.
Weir and C. C. Blackwell, eds., 1996); Gene Transfer Vectors for
Mammalian Cells (J. M. Miller and M. P. Calos, eds., 1987); PCR:
The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current
Protocols in Immunology (J. E. Coligan et al., eds., 1991); Short
Protocols in Molecular Biology (Ausubel et al., eds., J. Wiley and
Sons, 2002); Immunobiology (C. A. Janeway et al., 2004); Antibodies
(P. Finch, 1997); Antibodies: A Practical Approach (D. Catty., ed.,
IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach
(P. Shepherd and C. Dean, eds., Oxford University Press, 2000);
Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane, Cold
Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti
and J. D. Capra, eds., Harwood Academic Publishers, 1995); and
Cancer: Principles and Practice of Oncology (V. T. DeVita et al.,
eds., J.B. Lippincott Company, 2011).
Definitions
[0082] For purposes of interpreting this specification, the
following definitions will apply and whenever appropriate, terms
used in the singular will also include the plural and vice versa.
In the event that any definition set forth below conflicts with any
document incorporated herein by reference, the definition set forth
shall control.
[0083] As used herein, the singular form "a", "an", and "the"
includes plural references unless indicated otherwise.
[0084] It is understood that aspects and embodiments of the
invention described herein include "comprising," "consisting," and
"consisting essentially of" aspects and embodiments.
[0085] The term "about" as used herein refers to the usual error
range for the respective value readily known to the skilled person
in this technical field. Reference to "about" a value or parameter
herein includes (and describes) embodiments that are directed to
that value or parameter per se.
[0086] As used herein, a "peripheral blood mononuclear cells" or
"PBMCs" refers to a heterogeneous population of blood cells having
a round nucleus. Examples of cells that may be found in a
population of PBMCs include lymphocytes such as T cells, B cells,
NK cells (including NKT cells and CIK cells) and monocytes such as
macrophages and dendritic cells. A "plurality of PBMCs" as used
herein refers to a preparation of PBMCs comprising cells of at
least two types of blood cells. In some embodiments, a plurality of
PBMCs comprises two or more of T cells, B cells, NK cells,
macrophages or dendritic cells. In some embodiments, a plurality of
PBMCs comprises three or more of T cells, B cells, NK cells,
macrophages or dendritic cells. In some embodiments, a plurality of
PBMCs comprises four or more of T cells, B cells, NK cells,
macrophages or dendritic cells. In some embodiments, a plurality of
PBMCs comprises T cells, B cells, NK cells, macrophages and
dendritic cells.
[0087] PBMCs can be isolated by means known in the art. For
example, PBMCs can be derived from peripheral blood of an
individual based on density of PBMCs compared to other blood cells.
In some embodiments, PBMCs are derived from peripheral blood of an
individual using Ficoll (e.g., a ficoll gradient). In some
embodiments, PBMCs are derived from peripheral blood of an
individual using ELUTRA.RTM. cell separation system.
[0088] In some embodiments, a population of PBMCs is isolated from
an individual. In some embodiments, a plurality of PBMCs is an
autologous population of PBMCs where the population is derived from
a particular individual, manipulated by any of the methods
described herein, and returned to the particular individual. In
some embodiments, a plurality of PBMCs is an allogeneic population
of PBMCs where the population is derived from one individual,
manipulated by any of the methods described herein, and
administered to a second individual.
[0089] In some embodiments, a plurality of PBMCs is a reconstituted
preparation of PBMCs. In some embodiments, the plurality of PBMCs
may be generated by mixing cells typically found in a population of
PBMCs; for example, by mixing populations of two or more of T
cells, B cells, NK cells, or monocytes. In some embodiments, ratios
of cells in a population of splenocytes are adjusted (e.g.,
crafted) to better reflect the population profile of human PBMCs.
For example, B cells may be depleted from a population of
splenocytes to better reflect a population of human PBMCs.
[0090] The term "pore" as used herein refers to an opening,
including without limitation, a hole, tear, cavity, aperture,
break, gap, or perforation within a material. In some examples,
(where indicated) the term refers to a pore within a surface of the
present disclosure. In other examples, (where indicated) a pore can
refer to a pore in a cell membrane.
[0091] The term "membrane" as used herein refers to a selective
barrier or sheet containing pores. The term includes a pliable
sheet-like structure that acts as a boundary or lining. In some
examples, the term refers to a surface or filter containing pores.
This term is distinct from the term "cell membrane".
[0092] The term "filter" as used herein refers to a porous article
that allows selective passage through the pores. In some examples
the term refers to a surface or membrane containing pores.
[0093] The term "heterogeneous" as used herein refers to something
which is mixed or not uniform in structure or composition. In some
examples the term refers to pores having varied sizes, shapes or
distributions within a given surface.
[0094] The term "homogeneous" as used herein refers to something
which is consistent or uniform in structure or composition
throughout. In some examples the term refers to pores having
consistent sizes, shapes, or distribution within a given
surface.
[0095] The term "heterologous" as it relates to nucleic acid
sequences such as coding sequences and control sequences, denotes
sequences that are not normally joined together, and/or are not
normally associated with a particular cell. Thus, a "heterologous"
region of a nucleic acid construct or a vector is a segment of
nucleic acid within or attached to another nucleic acid molecule
that is not found in association with the other molecule in nature.
For example, a heterologous region of a nucleic acid construct
could include a coding sequence flanked by sequences not found in
association with the coding sequence in nature. Another example of
a heterologous coding sequence is a construct where the coding
sequence itself is not found in nature (e.g., synthetic sequences
having codons different from the native gene). Similarly, a cell
transformed with a construct which is not normally present in the
cell would be considered heterologous for purposes of this
invention. Allelic variation or naturally occurring mutational
events do not give rise to heterologous DNA, as used herein.
[0096] The term "heterologous" as it relates to amino acid
sequences such as peptide sequences and polypeptide sequences,
denotes sequences that are not normally joined together, and/or are
not normally associated with a particular cell. Thus, a
"heterologous" region of a peptide sequence is a segment of amino
acids within or attached to another amino acid molecule that is not
found in association with the other molecule in nature. For
example, a heterologous region of a peptide construct could include
the amino acid sequence of the peptide flanked by sequences not
found in association with the amino acid sequence of the peptide in
nature. Another example of a heterologous peptide sequence is a
construct where the peptide sequence itself is not found in nature
(e.g., synthetic sequences having amino acids different as coded
from the native gene). Similarly, a cell transformed with a vector
that expresses an amino acid construct which is not normally
present in the cell would be considered heterologous for purposes
of this invention. Allelic variation or naturally occurring
mutational events do not give rise to heterologous peptides, as
used herein.
[0097] The term "exogenous" when used in reference to an agent,
such as an antigen or an adjuvant, with relation to a cell refers
to an agent delivered from outside the cell (that is, from outside
the cell). The cell may or may not have the agent already present,
and may or may not produce the agent after the exogenous agent has
been delivered.
[0098] As used herein, the term "inhibit" may refer to the act of
blocking, reducing, eliminating, or otherwise antagonizing the
presence, or an activity of, a particular target. Inhibition may
refer to partial inhibition or complete inhibition. For example,
inhibiting an immune response may refer to any act leading to a
blockade, reduction, elimination, or any other antagonism of an
immune response. In other examples, inhibition of the expression of
a nucleic acid may include, but not limited to reduction in the
transcription of a nucleic acid, reduction of mRNA abundance (e.g.,
silencing mRNA transcription), degradation of mRNA, inhibition of
mRNA translation, and so forth.
[0099] As used herein, the term "suppress" may refer to the act of
decreasing, reducing, prohibiting, limiting, lessening, or
otherwise diminishing the presence, or an activity of, a particular
target. Suppression may refer to partial suppression or complete
suppression. For example, suppressing an immune response may refer
to any act leading to decreasing, reducing, prohibiting, limiting,
lessening, or otherwise diminishing an immune response. In other
examples, suppression of the expression of a nucleic acid may
include, but not limited to reduction in the transcription of a
nucleic acid, reduction of mRNA abundance (e.g., silencing mRNA
transcription), degradation of mRNA, inhibition of mRNA
translation, and so forth.
[0100] As used herein, the term "enhance" may refer to the act of
improving, boosting, heightening, or otherwise increasing the
presence, or an activity of, a particular target. For example,
enhancing an immune response may refer to any act leading to
improving, boosting, heightening, or otherwise increasing an immune
response. In one exemplary example, enhancing an immune response
may refer to employing an antigen and/or adjuvant to improve,
boost, heighten, or otherwise increase an immune response. In other
examples, enhancing the expression of a nucleic acid may include,
but not limited to increase in the transcription of a nucleic acid,
increase in mRNA abundance (e.g., increasing mRNA transcription),
decrease in degradation of mRNA, increase in mRNA translation, and
so forth.
[0101] As used herein, the term "modulate" may refer to the act of
changing, altering, varying, or otherwise modifying the presence,
or an activity of, a particular target. For example, modulating an
immune response may refer to any act leading to changing, altering,
varying, or otherwise modifying an immune response. In other
examples, modulating the expression of a nucleic acid may include,
but not limited to a change in the transcription of a nucleic acid,
a change in mRNA abundance (e.g., increasing mRNA transcription), a
corresponding change in degradation of mRNA, a change in mRNA
translation, and so forth.
[0102] As used herein, the term "induce" may refer to the act of
initiating, prompting, stimulating, establishing, or otherwise
producing a result. For example, inducing an immune response may
refer to any act leading to initiating, prompting, stimulating,
establishing, or otherwise producing a desired immune response. In
other examples, inducing the expression of a nucleic acid may
include, but not limited to initiation of the transcription of a
nucleic acid, initiation of mRNA translation, and so forth.
[0103] The term "homologous" as used herein refers to a molecule
which is derived from the same organism. In some examples the term
refers to a nucleic acid or protein which is normally found or
expressed within the given organism.
[0104] The term "polynucleotide" or "nucleic acid" as used herein
refers to a polymeric form of nucleotides of any length, either
ribonucleotides or deoxyribonucleotides. Thus, this term includes,
but is not limited to, single-, double- or multi-stranded DNA or
RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising
purine and pyrimidine bases, or other natural, chemically or
biochemically modified, non-natural, or derivatized nucleotide
bases. The backbone of the polynucleotide can comprise sugars and
phosphate groups (as may typically be found in RNA or DNA), or
modified or substituted sugar or phosphate groups. Alternatively,
the backbone of the polynucleotide can comprise a polymer of
synthetic subunits such as phosphoramidates and phosphorothioates,
and thus can be an oligodeoxynucleoside phosphoramidate (P--NH2) or
a mixed phosphoramidate-phosphodiester oligomer. In addition, a
double-stranded polynucleotide can be obtained from the single
stranded polynucleotide product of chemical synthesis either by
synthesizing the complementary strand and annealing the strands
under appropriate conditions, or by synthesizing the complementary
strand de novo using a DNA polymerase with an appropriate
primer.
[0105] The terms "polypeptide" and "protein" are used
interchangeably to refer to a polymer of amino acid residues, and
are not limited to a minimum length. Such polymers of amino acid
residues may contain natural or non-natural amino acid residues,
and include, but are not limited to, peptides, oligopeptides,
dimers, trimers, and multimers of amino acid residues. Both
full-length proteins and fragments thereof are encompassed by the
definition. The terms also include post-expression modifications of
the polypeptide, for example, glycosylation, sialylation,
acetylation, phosphorylation, and the like. Furthermore, for
purposes of the present invention, a "polypeptide" refers to a
protein which includes modifications, such as deletions, additions,
and substitutions (generally conservative in nature), to the native
sequence, as long as the protein maintains the desired activity.
These modifications may be deliberate, as through site-directed
mutagenesis, or may be accidental, such as through mutations of
hosts which produce the proteins or errors due to PCR
amplification.
[0106] As used herein, the term "adjuvant" refers to a substance
which either directly or indirectly modulates and/or engenders an
immune response. Generally, the adjuvant is administered in
conjunction with an antigen to effect enhancement of an immune
response to the antigen as compared to antigen alone. In some
embodiments, an adjuvant is used to condition a plurality of PBMCs
(e.g., as demonstrated in the Examples). Various adjuvants are
described herein.
[0107] The terms "CpG oligodeoxynucleotide" and "CpG ODN" refer to
DNA molecules containing a dinucleotide of cytosine and guanine
separated by a phosphate (also referred to herein as a "CpG"
dinucleotide, or "CpG"). The CpG ODNs of the present disclosure
contain at least one unmethylated CpG dinucleotide. That is, the
cytosine in the CpG dinucleotide is not methylated (i.e., is not
5-methylcytosine). CpG ODNs may have a partial or complete
phosphorothioate (PS) backbone.
[0108] As used herein, by "pharmaceutically acceptable" or
"pharmacologically compatible" is meant a material that is not
biologically or otherwise undesirable, e.g., the material may be
incorporated into a pharmaceutical composition administered to a
patient without causing any significant undesirable biological
effects or interacting in a deleterious manner with any of the
other components of the composition in which it is contained.
Pharmaceutically acceptable carriers or excipients have preferably
met the required standards of toxicological and manufacturing
testing and/or are included on the Inactive Ingredient Guide
prepared by the U.S. Food and Drug administration.
[0109] For any of the structural and functional characteristics
described herein, methods of determining these characteristics are
known in the art.
Modified PBMCs, Compositions, and Methods of Generating Modified
PBMCs
Modified PBMCs
[0110] In certain aspects, there is provided a plurality of
modified PBMCs comprising an antigen, wherein the antigen is
exogenous to the modified PBMCs. In other aspects, there is
provided a plurality of modified PMBCs comprising an antigen,
wherein the antigen is exogenous to the modified PBMCs, wherein the
antigen is a cancer antigen, an infectious disease antigen or a
viral-disease associated antigen. In some aspects, there is
provided a conditioned plurality of modified PBMCs comprising an
antigen, wherein the antigen is exogenous to the modified PBMCs. In
some aspects, there is provided a conditioned plurality of modified
PMBCs comprising an antigen, wherein the antigen is exogenous to
the modified PBMCs, wherein the antigen is a cancer antigen, an
infectious disease antigen or a viral-disease associated antigen.
In certain aspects, there is a conditioned plurality of modified
PBMCs comprising an antigen and an adjuvant, wherein the antigen is
exogenous to the modified PBMCs. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs. In some embodiments, the plurality of PBMCs
comprises a nucleic acid encoding an antigen. In some embodiments,
the plurality of PBMCs comprises an mRNA encoding an antigen. In
some embodiments, the one or more nucleic acids are carried in one
or more vehicles, wherein the one or more vehicles are delivered to
the input PBMCs. In some embodiments, the vehicle is a virus or a
viral-associated particle. In some embodiments, the virus comprises
one or more of: an adenovirus, an adeno-associated virus (AAV), a
baculovirus, a herpes virus, or a retrovirus. In some embodiments,
the virus comprises an AAV. In some embodiments, the vehicle is a
lipid-based vehicle, e.g., a liposome. In some embodiments, the
vehicle is a nanoparticle.
[0111] In some aspects, there is provided a plurality of modified
PBMCs comprising an antigen comprising the amino acid sequence of
any one of SEQ ID NOs: 18-25. In other aspects, there is provided a
conditioned plurality of modified PBMCs comprising an antigen
comprising the amino acid sequence of any one of SEQ ID NOs:
18-25.
[0112] In some aspects, there is provided a plurality of modified
PBMCs comprising an antigen comprising the amino acid sequence of
SEQ ID NO: 19. In other aspects, there is provided a conditioned
plurality of modified PBMCs comprising an antigen comprising the
amino acid sequence of SEQ ID NO: 19. In some aspects, there is
provided a plurality of modified PBMCs comprising an antigen
comprising the amino acid sequence of SEQ ID NO: 23. In other
aspects, there is provided a conditioned plurality of modified
PBMCs comprising an antigen comprising the amino acid sequence of
SEQ ID NO: 23.
[0113] In some aspects, there is provided a conditioned plurality
of PBMCs comprising an antigen, prepared by incubating the
plurality of PBMCs comprising the antigen with an adjuvant for a
sufficient time for the PBMCs to condition, thereby generating the
conditioned plurality of PBMCs comprising the antigen. In other
aspects, there is provided a conditioned plurality of PBMCs
comprising an antigen, prepared by incubating the plurality of
PBMCs with an adjuvant for a sufficient time for the PBMCs to
condition prior to introducing the antigen to the PBMCs, thereby
generating the conditioned plurality of PBMCs comprising the
antigen.
[0114] In some aspects, there is provided a conditioned plurality
of PBMCs comprising a nucleic acid (e.g., mRNA) encoding an
antigen, prepared by incubating the plurality of PBMCs comprising
the antigen with an adjuvant for a sufficient time for the PBMCs to
condition, thereby generating the conditioned plurality of PBMCs
comprising the antigen. In other aspects, there is provided a
conditioned plurality of PBMCs comprising a nucleic acid (e.g.,
mRNA) encoding an antigen, prepared by incubating the plurality of
PBMCs with an adjuvant for a sufficient time for the PBMCs to
condition prior to introducing the antigen to the PBMCs, thereby
generating the conditioned plurality of PBMCs comprising the
antigen.
[0115] Antigens and/or adjuvants can be introduced into PBMCs using
constriction-mediated delivery (SQZ). Therefore in some aspects,
there is provided a plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of: a) passing
a cell suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0116] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; and c) incubating the plurality of modified PBMCs
comprising the antigen with an adjuvant for a sufficient time for
the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the process further comprises
isolating the plurality of modified PBMCs comprising the antigen
from the cell suspension before incubation with the adjuvant to
condition the modified PBMCs. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs. In some embodiments, the one or more nucleic acids
are carried in one or more vehicles, wherein the one or more
vehicles are delivered to the input PBMCs. In some embodiments, the
vehicle is a virus or a viral-associated particle. In some
embodiments, the virus comprises one or more of: an adenovirus, an
adeno-associated virus (AAV), a baculovirus, a herpes virus, or a
retrovirus. In some embodiments, the virus comprises an AAV. In
some embodiments, the vehicle is a lipid-based vehicle, e.g., a
liposome. In some embodiments, the vehicle is a nanoparticle.
[0117] In some aspects, there is provided a plurality of modified
PBMCs comprising an antigen and an adjuvant, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen and the adjuvant to
pass through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the antigen
and the adjuvant for a sufficient time to allow the antigen and the
adjuvant to enter the perturbed input PBMCs; thereby generating the
plurality of modified PBMCs comprising the antigen and adjuvant. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0118] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) incubating a plurality of input PBMCs
with an adjuvant for a sufficient time for the input PBMCs to
condition, thereby generating a conditioned plurality of input
PBMCs; b) passing a cell suspension comprising the conditioned
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a conditioned plurality of perturbed input PBMCs; and c)
incubating the conditioned plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen. In some embodiments, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs.
[0119] In certain aspects, there is provided a plurality of
modified PBMCs comprising an antigen and an adjuvant, prepared by a
process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs comprising the adjuvant
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; and b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen and the
adjuvant. In other aspects, there is provided a plurality of
modified PBMCs comprising an antigen and an adjuvant, prepared by a
process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs comprising the antigen
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the adjuvant to pass through to form a plurality of
perturbed input PBMCs; and b) incubating the plurality of perturbed
input PBMCs with the adjuvant for a sufficient time to allow the
adjuvant to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen and the
adjuvant.
[0120] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0121] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising a human papillomavirus (HPV) antigen,
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the HPV antigen to pass through
to form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the HPV antigen for a
sufficient time to allow the HPV antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the HPV antigen; and c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, thereby generating the conditioned plurality of
modified PBMCs comprising the HPV antigen. In some embodiments, the
HPV antigen comprises one or more proteins. In some embodiments,
the HPV antigen is encoded by one or more nucleic acids and enters
the PBMC in the form of one or more nucleic acids, such as but not
limited to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments,
the HPV antigen is encoded by one or more mRNAs and enters the PBMC
in the form of one or more mRNAs.
[0122] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising a HPV antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the HPV antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the HPV antigen for a sufficient time to allow the
HPV antigen to enter the perturbed input PBMCs, thereby generating
a plurality of modified PBMCs comprising the HPV antigen; and
[0123] c) incubating the plurality of modified PBMCs comprising the
HPV antigen with a CpG ODN for a sufficient time for the modified
PBMCs comprising the HPV antigen to condition, wherein the CpG ODN
is CpG 7909, thereby generating the conditioned plurality of
modified PBMCs comprising the HPV antigen. In some embodiments, the
HPV antigen comprises one or more proteins. In some embodiments,
the HPV antigen is encoded by one or more nucleic acids and enters
the PBMC in the form of one or more nucleic acids, such as but not
limited to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments,
the HPV antigen is encoded by one or more mRNAs and enters the PBMC
in the form of one or more mRNAs.
[0124] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for about 1 hour to about 24 hours for the
modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0125] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising a HPV antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the HPV antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the HPV antigen for a sufficient time to allow the
HPV antigen to enter the perturbed input PBMCs, thereby generating
a plurality of modified PBMCs comprising the HPV antigen; and c)
incubating the plurality of modified PBMCs comprising the HPV
antigen with a CpG ODN for about 1 hour to about 24 hours for the
modified PBMCs comprising the HPV antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the HPV antigen. In some embodiments, the HPV antigen comprises one
or more proteins. In some embodiments, the HPV antigen is encoded
by one or more nucleic acids and enters the PBMC in the form of one
or more nucleic acids, such as but not limited to DNAs, cDNAs,
mRNAs, and plasmids. In some embodiments, the HPV antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0126] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising a HPV antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the HPV antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the HPV antigen for a sufficient time to allow the
HPV antigen to enter the perturbed input PBMCs, thereby generating
a plurality of modified PBMCs comprising the HPV antigen; and c)
incubating the plurality of modified PBMCs comprising the HPV
antigen with a CpG ODN for about 1 hour to about 24 hours for the
modified PBMCs comprising the HPV antigen to condition, wherein the
CpG ODN is CpG 7909, thereby generating the conditioned plurality
of modified PBMCs comprising the HPV antigen. In some embodiments,
the HPV antigen comprises one or more proteins. In some
embodiments, the HPV antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the HPV antigen is encoded by one or more mRNAs
and enters the PBMC in the form of one or more mRNAs.
[0127] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for about 1 hour to about 24 hours for the
modified PBMCs comprising the antigen to condition, wherein the
adjuvant is CpG 7909, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen. In some embodiments, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs.
[0128] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is (a) about 4.2 .mu.m to about 6 .mu.m; or (b) about
4.5 .mu.m. In some embodiments, the HPV antigen is incubated with a
CpG ODN for (a) about 2 hour to about 10 hours; (b) about 3 hours
to about 6 hours; or (c) about 4 hours.
[0129] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is about 3 .mu.m to about 6 .mu.m. In some
embodiments, the HPV antigen is incubated with a CpG ODN for (a)
about 2 hour to about 10 hours; (b) about 3 hours to about 6 hours;
or (c) about 4 hours.
[0130] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is (a) about 4.2 .mu.m to about 6 .mu.m; or (b) about
4.5 .mu.m. In some embodiments, the modified PBMCs comprising the
HPV antigen is incubated with a CpG ODN for (a) about 2 hour to
about 10 hours; (b) about 3 hours to about 6 hours; or (c) about 4
hours.
[0131] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is about 3 .mu.m to about 6 .mu.m. In some
embodiments, the modified PBMCs comprising the HPV antigen is
incubated with a CpG ODN for (a) about 2 hour to about 10 hours;
(b) about 3 hours to about 6 hours; or (c) about 4 hours.
[0132] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with a CpG ODN for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0133] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with a CpG ODN for a sufficient time for the modified PBMCs
comprising the antigen to condition, wherein the CpG ODN is CpG
7909, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0134] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with a CpG ODN for about 1 hour to about 24 hours for the modified
PBMCs comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0135] In some aspects, there is provided a conditioned plurality
of modified PBMCs comprising an antigen, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with a CpG ODN for about 1 hour to about 24 hours for the modified
PBMCs comprising the antigen to condition, wherein the CpG ODN is
CpG 7909, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0136] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is (a) about 4.2 .mu.m to about 6 .mu.m; or (b) about
4.5 .mu.m. In some embodiments, the antigen is incubated with a CpG
ODN for (a) about 2 hour to about 10 hours; (b) about 3 hours to
about 6 hours; or (c) about 4 hours.
[0137] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is about 3 .mu.m to about 6 .mu.m. In some
embodiments, the HPV antigen is incubated with a CpG ODN for (a)
about 2 hour to about 10 hours; (b) about 3 hours to about 6 hours;
or (c) about 4 hours.
[0138] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is (a) about 4.2 .mu.m to about 6 .mu.m; or (b) about
4.5 .mu.m. In some embodiments, the modified PBMCs comprising the
antigen is incubated with a CpG ODN for (a) about 2 hour to about
10 hours; (b) about 3 hours to about 6 hours; or (c) about 4
hours.
[0139] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the diameter of the
constriction is about 3 .mu.m to about 6 .mu.m. In some
embodiments, the modified PBMCs comprising the HPV antigen is
incubated with a CpG ODN for (a) about 2 hour to about 10 hours;
(b) about 3 hours to about 6 hours; or (c) about 4 hours.
[0140] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the concentration of the
antigen incubated with the perturbed input PBMCs is between about
0.1 .mu.M and about 1 mM and/or the concentration of the adjuvant
incubated with the perturbed input PBMCs is between about 0.1 .mu.M
and about 1 mM. In some embodiments, the concentration of the
antigen incubated with the perturbed input PBMCs is between about
0.1 .mu.M and about 10 .mu.M and/or the concentration of the
adjuvant incubated with the perturbed input PBMCs is between about
0.1 .mu.M and about 10 .mu.M. In some embodiments, the
concentration of the antigen incubated with the perturbed input
PBMCs is about 1 .mu.M and/or the concentration of the adjuvant
incubated with the perturbed input PBMCs is about 1 .mu.M. In some
embodiments, the ratio of the antigen to the adjuvant incubated
with the perturbed input PBMCs is between about 10000:1 to about
1:10000. In some embodiments, the ratio of the antigen to the
adjuvant incubated with the perturbed input PBMCs is about 200:1.
In some embodiments, the ratio of the antigen to the adjuvant
incubated with the perturbed input PBMCs is about 20:1. In some
embodiments, the antigen comprises one or more proteins. In some
embodiments, the antigen is encoded by one or more nucleic acids
and enters the PBMC in the form of one or more nucleic acids, such
as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In some
embodiments, the antigen is encoded by one or more mRNAs and enters
the PBMC in the form of one or more mRNAs.
[0141] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the process further comprises:
incubating the plurality of modified PBMCs comprising the antigen
and/or adjuvant with a second adjuvant for a sufficient time for
the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen and/or the adjuvant. In some embodiments, the process
further comprises isolating the plurality of modified PBMCs
comprising the antigen and/or the adjuvant from the cell suspension
before incubation with the adjuvant to condition the modified
PBMCs.
[0142] In some embodiments according to any one of the pluralities
of modified PBMCs described herein, the antigen is present in the
cytosol and the adjuvant is present in a vesicle of a cell in the
plurality of modified PBMCs. In some embodiments, the vesicle is an
endosome. In some embodiments, the antigen and/or the adjuvant are
present in multiple compartments of a cell in the plurality of
modified PBMCs. In further embodiments, the antigen and/or the
adjuvant are present in at least about 70% of the cells in the
plurality of PBMCs. In some embodiments, the antigen and/or the
adjuvant are present in at least any one of about 70%, about 75%,
about 80%, about 85%, about 95%, or about 99% of the cells in the
plurality of PBMCs. In some embodiments, the antigen is bound to
the surface of a cell in the plurality of modified PBMCs. In some
embodiments, the antigen and/or the adjuvant are present in at
least about 70% of cells of each of the T cells, B cells, NK cells,
and monocytes in the plurality of PBMCs. In some embodiments, the
antigen and/or the adjuvant are present in at least any one of
about 70%, about 75%, about 80%, about 85%, about 95%, or about 99%
of cells of each of the T cells, B cells, NK cells, and monocytes
in the plurality of PBMCs. In some embodiments, the antigen and/or
the adjuvant are present in at least about 70% of cells of one or
more of the T cells, B cells, NK cells, or monocytes in the
plurality of PBMCs. In some embodiments, the antigen and/or the
adjuvant are present in at least any one of about 70%, about 75%,
about 80%, about 85%, about 95%, or about 99% cells of one or more
of the T cells, B cells, NK cells, or monocytes in the plurality of
PBMCs.
[0143] In some embodiments, there is provided a composition
comprising any one of the pluralities of modified PBMCs described
herein. In some embodiments, there is provided a composition
comprising any one of the pluralities of modified PBMCs described
herein for use as a medicament. In some embodiments, there is
provided a composition comprising the any one of the pluralities of
modified PBMCs described herein for use in a method of treatment of
the human or animal body by surgery, therapy or diagnosis. In some
embodiments, there is provided a composition for use in treating a
cancer or an infectious disease comprising any one of the
pluralities of modified PBMCs described herein. In some
embodiments, there is provided a composition comprising any one of
the pluralities of modified PBMCs described herein for use in the
treatment of a cancer, an infectious disease or a viral-associated
disease. In some embodiments, the cancer is head and neck cancer,
cervical cancer, vulvar cancer, vaginal cancer, penile cancer, anal
cancer, perianal cancer, anogenital cancer, oral cancer or salivary
cancer. In some embodiments, the infectious disease is associated
with HIV, HPV, EBV, MCV, HBV or HCV. In some embodiments, there is
a pharmaceutical composition comprising any one of the pluralities
of modified PBMCs described herein, and a pharmaceutically
acceptable carrier. In some embodiments, the composition is for
treatment of cancers or infectious diseases.
[0144] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs. In some embodiments, the plurality of
PBMCs comprises a nucleic acid encoding an antigen. In some
embodiments, the plurality of PBMCs comprises an mRNA encoding an
antigen.
Compositions
[0145] In certain aspects, there is provided a composition
comprising a conditioned plurality of modified PBMCs comprising an
antigen for use as a medicament, wherein the conditioned plurality
of modified PBMCs is prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0146] In some aspects, there is provided a composition comprising
a conditioned plurality of modified PBMCs comprising an antigen for
use in a method of treatment of the human or animal body by
surgery, therapy or diagnosis, wherein the conditioned plurality of
modified PBMCs is prepared by a process comprising the steps of: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0147] In some aspects, there is provided a composition comprising
a conditioned plurality of modified PBMCs comprising an antigen for
use as a medicament, wherein the conditioned plurality of modified
PBMCs is prepared by a process comprising the steps of: a)
incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; and c) incubating the
conditioned plurality of perturbed input PBMCs with the antigen for
a sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0148] In some aspects, there is provided a composition comprising
a conditioned plurality of modified PBMCs comprising an antigen for
use in a method of treatment of the human or animal body, wherein
the conditioned plurality of modified PBMCs is prepared by a
process comprising the steps of: a) incubating a plurality of input
PBMCs with an adjuvant for a sufficient time for the input PBMCs to
condition, thereby generating a conditioned plurality of input
PBMCs; b) passing a cell suspension comprising the conditioned
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a conditioned plurality of perturbed input PBMCs; and c)
incubating the conditioned plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen. In some embodiments, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs.
[0149] In some aspects, there is provided a composition comprising
a conditioned plurality of modified PBMCs comprising an antigen for
use in a method of treating cancer an infectious disease or a viral
associated disease in an individual, wherein the conditioned
plurality of modified PBMCs is prepared by a process comprising the
steps of: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the antigen to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the antigen for a sufficient time to
allow the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen;
and c) incubating the plurality of modified PBMCs comprising the
antigen with an adjuvant for a sufficient time for the modified
PBMCs comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0150] In some aspects, there is provided a composition comprising
a conditioned plurality of modified PBMCs comprising an antigen for
use in the treatment of cancer, an infectious disease or a viral
associated disease in an individual, wherein the conditioned
plurality of modified PBMCs is prepared by a process comprising the
steps of: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the antigen to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the antigen for a sufficient time to
allow the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen;
and c) incubating the plurality of modified PBMCs comprising the
antigen with an adjuvant for a sufficient time for the modified
PBMCs comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0151] In some aspects, there is provided a composition comprising
a conditioned plurality of modified PBMCs comprising an antigen for
use in a method of treating a HPV-associated disease in an
individual, wherein the conditioned plurality of modified PBMCs is
prepared by a process comprising the steps of: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with an adjuvant for a
sufficient time for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0152] In some aspects, there is provided a composition comprising
a conditioned plurality of modified PBMCs comprising an antigen for
use in the treatment of a HPV-associated disease in an individual,
wherein the conditioned plurality of modified PBMCs is prepared by
a process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with an adjuvant for a sufficient time
for the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0153] In some aspects, there is provided a use of a composition
comprising a conditioned plurality of modified PBMCs comprising an
antigen in the manufacture of a medicament for treating cancer, an
infectious disease or a viral-associated disease in an individual,
wherein the conditioned plurality of modified PBMCs is prepared by
a process comprising the steps of: a) passing a cell suspension
comprising a plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with an adjuvant for a sufficient time
for the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0154] In some aspects, there is provided a use of a composition
comprising a conditioned plurality of modified PBMCs comprising an
antigen in the manufacture of a medicament for treating a
HPV-associated disease, wherein the conditioned plurality of
modified PBMCs is prepared by a process comprising the steps of: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0155] In some embodiments according to any one of the compositions
described herein, the concentration of the antigen incubated with
the perturbed input PBMCs is between about 0.1 .mu.M and about 1 mM
and/or the concentration of the adjuvant incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 1 mM. In
some embodiments, the concentration of the antigen incubated with
the perturbed input PBMCs is between about 0.1 .mu.M and about 10
.mu.M and/or the concentration of the adjuvant incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 10
.mu.M. In some embodiments, the concentration of the antigen
incubated with the perturbed input PBMCs is about 1 .mu.M and/or
the concentration of the adjuvant incubated with the perturbed
input PBMCs is about 1 .mu.M. In some embodiments, the ratio of the
antigen to the adjuvant incubated with the perturbed input PBMCs is
between about 10000:1 to about 1:10000. In some embodiments, the
ratio of the antigen to the adjuvant incubated with the perturbed
input PBMCs is about 200:1. In some embodiments, the ratio of the
antigen to the adjuvant incubated with the perturbed input PBMCs is
about 20:1. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0156] In some embodiments according to any one of the compositions
described herein, the process further comprises: incubating the
plurality of modified PBMCs comprising the antigen and/or adjuvant
with a second adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen
and/or the adjuvant. In some embodiments, the process further
comprises isolating the plurality of modified PBMCs comprising the
antigen and/or the adjuvant from the cell suspension before
incubation with the adjuvant to condition the modified PBMCs.
[0157] In some embodiments according to any one of the compositions
described herein, the antigen is present in the cytosol and the
adjuvant is present in a vesicle of a cell in the plurality of
modified PBMCs. In some embodiments, the vesicle is an endosome. In
some embodiments, the antigen and/or the adjuvant are present in
multiple compartments of a cell in the plurality of modified PBMCs.
In further embodiments, the antigen and/or the adjuvant are present
in at least about 70% of the cells in the plurality of PBMCs. In
some embodiments, the antigen and/or the adjuvant are present in at
least any one of about 70%, about 75%, about 80%, about 85%, about
95%, or about 99%, or 100% of the cells in the plurality of PBMCs.
In some embodiments, the antigen is bound to the surface of a cell
in the plurality of modified PBMCs.
[0158] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs. In some embodiments, the plurality of
PBMCs comprises a nucleic acid encoding an antigen. In some
embodiments, the plurality of PBMCs comprises an mRNA encoding an
antigen.
Methods of Generating a Plurality of Modified PBMCs
[0159] In some aspects, also provided is a method for generating a
conditioned plurality of PBMCs comprising an antigen, comprising
incubating a plurality of PBMCs comprising the antigen with an
adjuvant for a sufficient time for the PBMCs to condition, thereby
generating the conditioned plurality of PBMCs comprising the
antigen.
[0160] In some aspects, there is a method for generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the antigen to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the antigen for a sufficient time to
allow the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen;
and c) incubating the plurality of modified PBMCs comprising the
antigen with an adjuvant for a sufficient time for the modified
PBMCs comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0161] In some aspects, there is provided a method for generating a
plurality of modified PBMCs comprising an antigen, comprising: a)
passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; and b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen. In some
embodiments, the antigen comprises one or more proteins. In some
embodiments, the antigen is encoded by one or more nucleic acids
and enters the PBMC in the form of one or more nucleic acids, such
as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In some
embodiments, the antigen is encoded by one or more mRNAs and enters
the PBMC in the form of one or more mRNAs.
[0162] In some aspects, there is provided a method for generating a
plurality of modified PBMCs comprising an antigen and an adjuvant,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the antigen and the adjuvant to pass
through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the antigen
and the adjuvant for a sufficient time to allow the antigen and the
adjuvant to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen and adjuvant. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0163] In some aspects, there is provided a method of generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) incubating a plurality of input PBMCs with an
adjuvant for a sufficient time for the input PBMCs to condition,
thereby generating a conditioned plurality of input PBMCs; b)
passing a cell suspension comprising the conditioned plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for the antigen to pass through to form a
conditioned plurality of perturbed input PBMCs; and c) incubating
the conditioned plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating the conditioned plurality of
modified PBMCs comprising the antigen. In some embodiments, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs.
[0164] In certain aspects, there is provided a method for
generating a plurality of modified PBMCs comprising an antigen and
an adjuvant, comprising: a) passing a cell suspension comprising a
plurality of input PBMCs comprising an adjuvant through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
an antigen to pass through to form a plurality of perturbed input
PBMCs; and b) incubating the plurality of perturbed input PBMCs
with the antigen for a sufficient time to allow the antigen to
enter the perturbed input PBMCs, thereby generating the plurality
of modified PBMCs comprising the antigen and the adjuvant. In some
embodiments, the antigen comprises one or more proteins. In some
embodiments, the antigen is encoded by one or more nucleic acids
and enters the PBMC in the form of one or more nucleic acids, such
as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In some
embodiments, the antigen is encoded by one or more mRNAs and enters
the PBMC in the form of one or more mRNAs.
[0165] In some aspects, there is provided a method for generating a
plurality of modified PBMCs comprising an antigen and an adjuvant,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs comprising an antigen through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for an adjuvant to
pass through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the adjuvant
for a sufficient time to allow the adjuvant to enter the perturbed
input PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen and the adjuvant.
[0166] In certain aspects, there is provided a method for
generating a conditioned plurality of modified PBMCs comprising an
antigen, comprising: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is about 3 .mu.m to about 10
.mu.m, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0167] In certain aspects, there is provided a method for
generating a conditioned plurality of modified PBMCs comprising a
human papillomavirus (HPV) antigen, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the HPV antigen to pass through
to form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the HPV antigen for a
sufficient time to allow the HPV antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the HPV antigen; and c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, thereby generating the conditioned plurality of
modified PBMCs comprising the HPV antigen. In some embodiments, the
HPV antigen comprises one or more proteins. In some embodiments,
the HPV antigen is encoded by one or more nucleic acids and enters
the PBMC in the form of one or more nucleic acids, such as but not
limited to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments,
the HPV antigen is encoded by one or more mRNAs and enters the PBMC
in the form of one or more mRNAs.
[0168] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an HPV antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; and c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for a sufficient time for the modified PBMCs comprising the HPV
antigen to condition, wherein the CpG ODN is CpG 7909, thereby
generating the conditioned plurality of modified PBMCs comprising
the HPV antigen. In some embodiments, the HPV antigen comprises one
or more proteins. In some embodiments, the HPV antigen is encoded
by one or more nucleic acids and enters the PBMC in the form of one
or more nucleic acids, such as but not limited to DNAs, cDNAs,
mRNAs, and plasmids. In some embodiments, the HPV antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0169] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an HPV antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; and c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for about 1 hour to about 24 hours for the modified PBMCs
comprising the HPV antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the HPV antigen.
In some embodiments, the HPV antigen comprises one or more
proteins. In some embodiments, the HPV antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the HPV antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0170] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an HPV antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; and c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for about 1 hour to about 24 hours for the modified PBMCs
comprising the HPV antigen to condition, wherein the CpG ODN is CpG
7909, thereby generating the conditioned plurality of modified
PBMCs comprising the HPV antigen. In some embodiments, the HPV
antigen comprises one or more proteins. In some embodiments, the
HPV antigen is encoded by one or more nucleic acids and enters the
PBMC in the form of one or more nucleic acids, such as but not
limited to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments,
the HPV antigen is encoded by one or more mRNAs and enters the PBMC
in the form of one or more mRNAs.
[0171] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with an adjuvant for about 1
hour to about 24 hours for the modified PBMCs comprising the
antigen to condition, wherein the adjuvant is CpG 7909, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0172] In some embodiments according to any one of the methods
described herein, the diameter of the constriction is (a) about 4.2
.mu.m to about 6 .mu.m; or (b) about 4.5 .mu.m. In some
embodiments, the diameter of the constriction is about 3 .mu.m to
about 6 .mu.m. In some embodiments, the plurality of modified PBMCs
comprising the HPV antigen is incubated with a CpG ODN for (a)
about 2 hour to about 10 hours; (b) about 3 hours to about 6 hours;
or (c) about 4 hours.
[0173] In some embodiments according to any one of the methods
described herein, the diameter of the constriction is (a) about 4.2
.mu.m to about 6 .mu.m; or (b) about 4.5 .mu.m. In some
embodiments, the diameter of the constriction is about 3 .mu.m to
about 6 .mu.m. In some embodiments, the plurality of modified PBMCs
comprising the antigen is incubated with an adjuvant for (a) about
2 hour to about 10 hours; (b) about 3 hours to about 6 hours; or
(c) about 4 hours.
[0174] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0175] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the antigen to
condition, wherein the CpG ODN is CpG 7909, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0176] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with a CpG ODN for about 1
hour to about 24 hours for the modified PBMCs comprising the
antigen to condition, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen. In some embodiments, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs.
[0177] In some aspects, there is provided a method for generating a
conditioned plurality of modified PBMCs comprising an antigen,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with a CpG ODN for about 1
hour to about 24 hours for the modified PBMCs comprising the
antigen to condition, wherein the CpG ODN is CpG 7909, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0178] In some embodiments according to any one of the methods
described herein, the diameter of the constriction is (a) about 4.2
.mu.m to about 6 .mu.m; or (b) about 4.5 .mu.m. In some
embodiments, the diameter of the constriction is about 3 .mu.m to
about 6 .mu.m. In some embodiments, the plurality of modified PBMCs
comprising the antigen is incubated with a CpG ODN for (a) about 2
hour to about 10 hours; (b) about 3 hours to about 6 hours; or (c)
about 4 hours.
[0179] In some embodiments according to any one of the methods
described herein, the diameter of the constriction is (a) about 4.2
.mu.m to about 6 .mu.m; or (b) about 4.5 .mu.m. In some
embodiments, the diameter of the constriction is about 3 .mu.m to
about 6 .mu.m. In some embodiments, the plurality of modified PBMCs
comprising the antigen is incubated with an adjuvant for (a) about
2 hour to about 10 hours; (b) about 3 hours to about 6 hours; or
(c) about 4 hours.
[0180] In some embodiments according to any one of the methods
described herein, the concentration of the antigen incubated with
the perturbed input PBMCs is between about 0.1 .mu.M and about 1 mM
and/or the concentration of the adjuvant incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 1 mM. In
some embodiments, the concentration of the antigen incubated with
the perturbed input PBMCs is between about 0.1 .mu.M and about 10
.mu.M and/or the concentration of the adjuvant incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 10
.mu.M. In some embodiments, the concentration of the antigen
incubated with the perturbed input PBMCs is about 1 .mu.M and/or
the concentration of the adjuvant incubated with the perturbed
input PBMCs is about 1 .mu.M. In some embodiments, the ratio of the
antigen to the adjuvant incubated with the perturbed input PBMCs is
between about 10000:1 to about 1:10000. In some embodiments, the
ratio of the antigen to the adjuvant incubated with the perturbed
input PBMCs is about 200:1. In some embodiments, the ratio of the
antigen to the adjuvant incubated with the perturbed input PBMCs is
about 20:1.
[0181] In some embodiments according to any one of the methods
described herein, the process further comprises: incubating the
plurality of modified PBMCs comprising the antigen and/or adjuvant
with a second adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen
and/or the adjuvant. In some embodiments, the process further
comprises isolating the plurality of modified PBMCs comprising the
antigen and/or the adjuvant from the cell suspension before
incubation with the adjuvant to condition the modified PBMCs.
[0182] In some embodiments according to any one of the methods
described herein, the antigen is present in the cytosol and the
adjuvant is present in a vesicle of a cell in the plurality of
modified PBMCs. In some embodiments, the vesicle is an endosome. In
some embodiments, the antigen and/or the adjuvant are present in
multiple compartments of a cell in the plurality of modified PBMCs.
In further embodiments, the antigen and/or the adjuvant are present
in at least about 70% of the cells in the plurality of PBMCs. In
some embodiments, the antigen and/or the adjuvant are present in at
least any one of about 70%, about 75%, about 80%, about 85%, about
95%, or about 99%, or 100% of the cells in the plurality of PBMCs.
In some embodiments, the antigen is bound to the surface of a cell
in the plurality of modified PBMCs. In some embodiments, the
antigen and/or the adjuvant are present in at least any one of
about 70%, about 75%, about 80%, about 85%, about 95%, or about
99%, or 100% of cells of each of the T cells, B cells, NK cells,
and monocytes within the plurality of modified PBMCs. In some
embodiments, the antigen and/or the adjuvant are present in at
least any one of about 70%, about 75%, about 80%, about 85%, about
95%, or about 99%, or 100% of cells of one or more of the T cells,
B cells, NK cells, or monocytes within the plurality of modified
PBMCs.
[0183] In some embodiments according to any one of the methods
described herein, the process further comprises a step of
incubating the input PBMCs and/or the modified PBMCs with an agent
that enhances the viability and/or function of the modified PBMCs
as compared to corresponding modified PBMCs prepared without the
further incubation step.
Methods of Stimulating a Response in an Individual
[0184] In some aspects, the present invention provides methods for
treating and preventing a cancer or an infectious disease, and/or
modulating the immune response in an individual with a cancer or an
infectious disease comprising administering to the individual a
composition comprising a plurality of modified PBMCs, wherein the
modified PBMCs comprise intracellularly an antigen associated with
cancer or with an infectious disease.
[0185] In some embodiments, there is provided a method for
stimulating an immune response in an individual, comprising
administering to the individual any one of the pluralities of
modified PBMCs, compositions, or pharmaceutical compositions
described herein.
[0186] In certain aspects, there is provided a method for
stimulating an immune response in an individual, comprising: a)
administering a plurality of modified PBMCs comprising an antigen
comprising the amino acid sequence of any one of SEQ ID NOs: 18-25
to the individual; and b) administering an adjuvant to the
individual.
[0187] In certain aspects, there is provided a method for
stimulating an immune response in an individual, comprising: a)
incubating a plurality of PBMCs comprising an antigen with an
adjuvant for a sufficient time for the PBMCs to condition, thereby
generating a conditioned plurality of PBMCs comprising the antigen;
b) administering the conditioned plurality of PBMCs comprising the
antigen to the individual.
[0188] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) incubating a
plurality of PBMCs with an adjuvant for a sufficient time for the
PBMCs to condition, thereby generating a conditioned plurality of
PBMCs comprising the antigen; b) introducing an antigen to the
plurality of PBMCs; and c) administering the conditioned plurality
of PBMCs comprising the antigen to the individual.
[0189] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
an antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; c) incubating the plurality of
modified PBMCs comprising the antigen with an adjuvant for a
sufficient time for the modified PBMCs comprising the antigen to
condition, thereby generating a conditioned plurality of modified
PBMCs comprising the antigen; and d) administering the conditioned
plurality of modified PBMCs comprising the antigen to the
individual. In some embodiments, the method further comprises
isolating the plurality of modified PBMCs comprising the antigen
from the cell suspension before incubation with the adjuvant. In
some embodiments, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
[0190] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
an antigen and an adjuvant to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen and the adjuvant for a sufficient time
to allow the antigen and the adjuvant to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen and adjuvant; and c) administering the plurality of
modified PBMCs to the individual. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0191] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) incubating a
plurality of input PBMCs with an adjuvant for a sufficient time for
the input PBMCs to condition, thereby generating a conditioned
plurality of input PBMCs; b) passing a cell suspension comprising
the conditioned plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for an antigen to
pass through to form a conditioned plurality of perturbed input
PBMCs; c) incubating the conditioned plurality of perturbed input
PBMCs with the antigen for a sufficient time to allow the antigen
to enter the perturbed input PBMCs, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen; and
d) administering the conditioned plurality of modified PBMCs to the
individual. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0192] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs comprising an
adjuvant through a cell-deforming constriction, wherein a diameter
of the constriction is a function of a diameter of the input PBMCs
in the suspension, thereby causing perturbations of the input PBMCs
large enough for an antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen and the
adjuvant; and c) administering the plurality of modified PBMCs to
the individual. In some embodiments, the antigen comprises one or
more proteins. In some embodiments, the antigen is encoded by one
or more nucleic acids and enters the PBMC in the form of one or
more nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs,
and plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0193] In certain aspects, there is provided a method for
stimulating an immune response in an individual, comprising: a)
passing a cell suspension comprising an input PBMCs comprising an
antigen through a cell-deforming constriction, wherein a diameter
of the constriction is a function of a diameter of the input PBMCs
in the suspension, thereby causing perturbations of the input PBMCs
large enough for an adjuvant to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the adjuvant for a sufficient time to allow the
adjuvant to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen and the
adjuvant; and c) administering the plurality of modified PBMCs to
the individual. In some embodiments, the antigen comprises one or
more proteins. In some embodiments, the antigen is encoded by one
or more nucleic acids and enters the PBMC in the form of one or
more nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs,
and plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0194] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is a function of a diameter of the input PBMCs in the suspension,
thereby causing perturbations of the input PBMCs large enough for
an antigen to pass through to form a plurality of perturbed input
PBMCs; b) incubating the plurality of perturbed input PBMCs with
the antigen for a sufficient time to allow the antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen; c) administering the plurality of
modified PBMCs to the individual; and d) administering an adjuvant
to the individual. In some embodiments, the antigen comprises one
or more proteins. In some embodiments, the antigen is encoded by
one or more nucleic acids and enters the PBMC in the form of one or
more nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs,
and plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0195] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising an input PBMCs comprising an antigen through
a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an adjuvant to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the adjuvant for a sufficient time to allow the
adjuvant to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen and the
adjuvant; and c) administering the plurality of modified PBMCs to
the individual; and d) administering an adjuvant to the
individual.
[0196] In some aspects, there is provided a method for stimulating
an immune response against an HPV antigen in an individual,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for a sufficient time for the modified PBMCs comprising the HPV
antigen to condition, thereby generating the conditioned plurality
of modified PBMCs comprising the HPV antigen; and d) administering
the conditioned plurality of modified PBMCs comprising the HPV
antigen to the individual. In some embodiments, the antigen
comprises one or more proteins. In some embodiments, the antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0197] In some aspects, there is provided a method for stimulating
an immune response against an HPV antigen in an individual,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for a sufficient time for the modified PBMCs comprising the HPV
antigen to condition, wherein the CpG ODN is CpG 7909, thereby
generating the conditioned plurality of modified PBMCs comprising
the HPV antigen; and d) administering the conditioned plurality of
modified PBMCs comprising the HPV antigen to the individual. In
some embodiments, the HPV antigen comprises one or more proteins.
In some embodiments, the HPV antigen is encoded by one or more
nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the HPV antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more mRNAs
[0198] In some aspects, there is provided a method for stimulating
an immune response against an HPV antigen in an individual,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for about 1 hour to about 24 hours for the modified PBMCs
comprising the HPV antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the HPV antigen;
and d) administering the conditioned plurality of modified PBMCs
comprising the HPV antigen to the individual. In some embodiments,
the HPV antigen comprises one or more proteins. In some
embodiments, the HPV antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the HPV antigen is encoded by one or more mRNAs
and enters the PBMC in the form of one or more mRNAs
[0199] In some aspects, there is provided a method for stimulating
an immune response against an HPV antigen in an individual,
comprising: a) passing a cell suspension comprising a plurality of
input PBMCs through a cell-deforming constriction, wherein a
diameter of the constriction is about 3 .mu.m to about 10 .mu.m,
thereby causing perturbations of the input PBMCs large enough for
the HPV antigen to pass through to form a plurality of perturbed
input PBMCs; b) incubating the plurality of perturbed input PBMCs
with the HPV antigen for a sufficient time to allow the HPV antigen
to enter the perturbed input PBMCs, thereby generating a plurality
of modified PBMCs comprising the HPV antigen; c) incubating the
plurality of modified PBMCs comprising the HPV antigen with a CpG
ODN for about 1 hour to about 24 hours for the modified PBMCs
comprising the HPV antigen to condition, wherein the CpG ODN is CpG
7909, thereby generating the conditioned plurality of modified
PBMCs comprising the HPV antigen; and d) administering the
conditioned plurality of modified PBMCs comprising the HPV antigen
to the individual. In some embodiments, the HPV antigen comprises
one or more proteins. In some embodiments, the HPV antigen is
encoded by one or more nucleic acids and enters the PBMC in the
form of one or more nucleic acids, such as but not limited to DNAs,
cDNAs, mRNAs, and plasmids. In some embodiments, the HPV antigen is
encoded by one or more mRNAs and enters the PBMC in the form of one
or more mRNAs.
[0200] In some embodiments according to any one of the methods
described herein, the diameter of the constriction is about 4 .mu.m
to about 10 .mu.m. In some embodiments, the diameter of the
constriction is about 3 .mu.m to about 6 .mu.m. In some
embodiments, the diameter of the constriction is (a) about 4.2
.mu.m to about 6 .mu.m; or (b) about 4.5 .mu.m. In some
embodiments, the plurality of modified PBMCs comprising the HPV
antigen is incubated with a CpG ODN for (a) about 2 hour to about
10 hours; (b) about 3 hours to about 6 hours; or (c) about 4
hours.
[0201] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for an antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for a sufficient time for the
modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen; and d) administering the conditioned plurality of
modified PBMCs comprising the antigen to the individual. In some
embodiments, the antigen comprises one or more proteins. In some
embodiments, the antigen is encoded by one or more nucleic acids
and enters the PBMC in the form of one or more nucleic acids, such
as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In some
embodiments, the antigen is encoded by one or more mRNAs and enters
the PBMC in the form of one or more mRNAs.
[0202] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for an antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for a sufficient time for the
modified PBMCs comprising the antigen to condition, wherein the CpG
ODN is CpG 7909, thereby generating the conditioned plurality of
modified PBMCs comprising the antigen; and d) administering the
conditioned plurality of modified PBMCs comprising the antigen to
the individual. In some embodiments, the antigen comprises one or
more proteins. In some embodiments, the antigen is encoded by one
or more nucleic acids and enters the PBMC in the form of one or
more nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs,
and plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0203] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for about 1 hour to about 24
hours for the modified PBMCs comprising the antigen to condition,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen; and d) administering the conditioned
plurality of modified PBMCs comprising the antigen to the
individual. In some embodiments, the antigen comprises one or more
proteins. In some embodiments, the antigen is encoded by one or
more nucleic acids and enters the PBMC in the form of one or more
nucleic acids, such as but not limited to DNAs, cDNAs, mRNAs, and
plasmids. In some embodiments, the antigen is encoded by one or
more mRNAs and enters the PBMC in the form of one or more
mRNAs.
[0204] In some aspects, there is provided a method for stimulating
an immune response in an individual, comprising: a) passing a cell
suspension comprising a plurality of input PBMCs through a
cell-deforming constriction, wherein a diameter of the constriction
is about 3 .mu.m to about 10 .mu.m, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen; c) incubating the plurality of modified PBMCs
comprising the antigen with a CpG ODN for about 1 hour to about 24
hours for the modified PBMCs comprising the antigen to condition,
wherein the CpG ODN is CpG 7909, thereby generating the conditioned
plurality of modified PBMCs comprising the antigen; and d)
administering the conditioned plurality of modified PBMCs
comprising the antigen to the individual. In some embodiments, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs.
[0205] In some embodiments according to any one of the methods
described herein, the diameter of the constriction is about 4 .mu.m
to about 10 .mu.m. In some embodiments, the diameter of the
constriction is about 3 .mu.m to about 6 .mu.m. In some
embodiments, the diameter of the constriction is (a) about 4.2
.mu.m to about 6 .mu.m; or (b) about 4.5 .mu.m. In some
embodiments, the plurality of modified PBMCs comprising the antigen
is incubated with a CpG ODN for (a) about 2 hour to about 10 hours;
(b) about 3 hours to about 6 hours; or (c) about 4 hours.
[0206] In some embodiments according to any one of the methods
described herein, the concentration of the antigen incubated with
the perturbed input PBMCs is between about 0.1 .mu.M and about 1 mM
and/or the concentration of the adjuvant incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 1 mM. In
some embodiments, the concentration of the antigen incubated with
the perturbed input PBMCs is between about 0.1 .mu.M and about 10
.mu.M and/or the concentration of the adjuvant incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 10
.mu.M. In some embodiments, the concentration of the antigen
incubated with the perturbed input PBMCs is about 1 .mu.M and/or
the concentration of the adjuvant incubated with the perturbed
input PBMCs is about 1 .mu.M. In some embodiments, the ratio of the
antigen to the adjuvant incubated with the perturbed input PBMCs is
between about 10000:1 to about 1:10000. In some embodiments, the
ratio of the antigen to the adjuvant incubated with the perturbed
input PBMCs is about 200:1. In some embodiments, the ratio of the
antigen to the adjuvant incubated with the perturbed input PBMCs is
about 20:1.
[0207] In some embodiments according to any one of the methods
described herein, the process further comprises: incubating the
plurality of modified PBMCs comprising the antigen and/or adjuvant
with a second adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen
and/or the adjuvant. In some embodiments, the process further
comprises isolating the plurality of modified PBMCs comprising the
antigen and/or the adjuvant from the cell suspension before
incubation with the adjuvant to condition the modified PBMCs.
[0208] In some embodiments according to any one of the methods
described herein, the antigen is present in the cytosol and the
adjuvant is present in a vesicle of a cell in the plurality of
modified PBMCs. In some embodiments, the vesicle is an endosome. In
some embodiments, the antigen and/or the adjuvant are present in
multiple compartments of a cell in the plurality of modified PBMCs.
In further embodiments, the antigen and/or the adjuvant are present
in at least about 70% of the cells in the plurality of PBMCs. In
some embodiments, the antigen and/or the adjuvant are present in at
least any one of about 70%, about 75%, about 80%, about 85%, about
95%, or about 99%, or 100% of the cells in the plurality of PBMCs.
In some embodiments, the antigen is bound to the surface of a cell
in the plurality of modified PBMCs. In some embodiments, the
antigen is presented in at least any one of about 50%, about 60%,
about 70%, about 75%, about 80%, about 85%, about 95%, about 99%,
or 100% of the cells in the plurality of modified PBMCs. In some
embodiments, the antigen is presented in at least any one of about
50%, about 60%, about 70%, about 75%, about 80%, about 85%, about
95%, about 99%, or 100% of cells in one or more of the T cells, B
cells, NK cells, or monocytes within the plurality of modified
PBMCs. In some embodiments, the antigen is presented in at least
any one of about 50%, about 60%, about 70%, about 75%, about 80%,
about 85%, about 95%, about 99%, or 100% of cells of each of the T
cells, B cells, NK cells, and monocytes within the plurality of
modified PBMCs. In some embodiments, the antigen is presented in at
least any one of about 50%, about 60%, about 70%, about 75%, about
80%, about 85%, about 95%, about 99%, or 100% of cells of one or
more of the T cells, B cells, NK cells, and monocytes within the
plurality of modified PBMCs.
[0209] In some embodiments according to any one of the methods
described herein, the process further comprises a step of
incubating the input PBMCs and/or the modified PBMCs with an agent
that enhances the viability and/or function of the modified PBMCs
as compared to corresponding modified PBMCs prepared without the
further incubation step.
[0210] In some embodiments according to any one of the methods
described herein, the method further comprises administering a
third adjuvant to the individual. In some embodiments, the
composition comprising the plurality of modified PBMCs and the
third adjuvant are administered simultaneously. In some
embodiments, the third adjuvant is administered before,
concurrently with, or after administration of the plurality of
modified PBMCs to the individual. In some embodiments, the
composition comprising the plurality of modified PBMCs and the
third adjuvant are administered sequentially. In some embodiments,
the third adjuvant is the same as the constriction-delivered
adjuvant. In some embodiments, the third adjuvant is the same as
the conditioning adjuvant. In some embodiments, the third adjuvant
is the different from the constriction-delivered adjuvant. In some
embodiments, the third adjuvant is different from the conditioning
adjuvant.
[0211] In some embodiments, the method comprises multiple
administrations of the modified PBMCs. In some embodiments, the
method comprises about 3 to about 9 administrations of the modified
PBMCs. In some embodiments, the method comprises about any one of
1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 administrations
of the modified PBMCs. In some embodiments, the method comprises
continuous administrations of the modified PBMCs as needed. IN some
embodiments, the time interval between two successive
administrations of the plurality of modified PBMCs is between about
1 day and about 30 days. In some embodiments, the time interval
between two successive administrations of the plurality of modified
PBMCs is about 21 days. In some embodiments, the time the time
interval between two successive administrations of the modified
immune cells is about any one of 1, 2, 3, 4, 5, 6, 7, 8, 10, 12,
14, 16, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90,
95, 100, or 150 days. In some embodiments, the individual is
positive for expression of HLA-A2. In some embodiments, at least
one cell in the plurality of modified PBMCs is positive for
expression of HLA-A2. In some embodiments, at least about any one
of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of the
modified PBMCs is positive for expression of HLA-A2. In some
embodiments, at least about any one of 10%, 20%, 30%, 40%, 50%,
60%, 70%, 80%, 90%, or 99% of T cells within the modified PBMCs are
positive for expression of HLA-A2. In some embodiments, at least
about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or
99% of B cells within the modified PBMCs are positive for
expression of HLA-A2. In some embodiments, at least about any one
of 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 99% of NK cells
within the modified PBMCs are positive for expression of HLA-A2. In
some embodiments, at least about any one of 10%, 20%, 30%, 40%,
50%, 60%, 70%, 80%, 90%, or 99% of monocytes within the modified
PBMCs are positive for expression of HLA-A2.
[0212] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered prior to administering
the third adjuvant. For example, the composition comprising the
plurality of modified PBMCs is administered from about 1 hour to
about 1 week prior to administration of the third adjuvant. For
example, in some embodiments, the composition comprising the
plurality of modified PBMCs is administered about 1 hour, about 2
hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours,
about 10 hours, about 12 hours, about 14 hours, about 16 hours,
about 18 hours, about 20 hours, about 24 hours, about 30 hours,
about 36 hours, about 42 hours, about 48 hours, about 60 hours,
about 3 days, about 4 days, about 5 days, about 6 days, or about 7
days prior to administration of the third adjuvant. In some
embodiments, the composition comprising the plurality of modified
PBMCs is administered from between about 1 hour and about 2 hours,
from between about 2 hours and about 3 hours, from between about 3
hours and about 4 hours, from between about 4 hours and about 6
hours, from between about 6 hours and about 8 hours, from between
about 8 hours and about 10 hours, from between about 10 hours and
about 12 hours, from between about 12 hours and about 14 hours,
from between about 14 hours and about 16 hours, from between about
16 hours and about 18 hours, from between about 18 hours and about
20 hours, from between about 20 hours and about 24 hours, from
between about 24 hours and about 30 hours, from between about 30
hours and about 36 hours, from between about 36 hours and about 42
hours, from between about 42 hours and about 48 hours, from between
about 48 hours and about 60 hours, from between about 60 hours and
about 3 days, from between about 3 days and about 4 days, from
between about 4 days and about 5 days, from between about 5 days
and about 6 days, from between about 6 days and about 7 days prior
to administration of the third adjuvant.
[0213] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered following
administration of the third adjuvant. For example, the composition
comprising the plurality of modified PBMCs is administered from
about 1 hour to about 1 week following administration of the third
adjuvant. For example, in some embodiments, the composition
comprising the plurality of modified PBMCs is administered about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours,
about 8 hours, about 10 hours, about 12 hours, about 14 hours,
about 16 hours, about 18 hours, about 20 hours, about 24 hours,
about 30 hours, about 36 hours, about 42 hours, about 48 hours,
about 60 hours, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days following administration of the third
adjuvant. In some embodiments, the composition comprising the
plurality of modified PBMCs is administered from between about 1
hour and about 2 hours, from between about 2 hours and about 3
hours, from between about 3 hours and about 4 hours, from between
about 4 hours and about 6 hours, from between about 6 hours and
about 8 hours, from between about 8 hours and about 10 hours, from
between about 10 hours and about 12 hours, from between about 12
hours and about 14 hours, from between about 14 hours and about 16
hours, from between about 16 hours and about 18 hours, from between
about 18 hours and about 20 hours, from between about 20 hours and
about 24 hours, from between about 24 hours and about 30 hours,
from between about 30 hours and about 36 hours, from between about
36 hours and about 42 hours, from between about 42 hours and about
48 hours, from between about 48 hours and about 60 hours, from
between about 60 hours and about 3 days, from between about 3 days
and about 4 days, from between about 4 days and about 5 days, from
between about 5 days and about 6 days, from between about 6 days
and about 7 days following administration of the third
adjuvant.
[0214] In some embodiments, the third adjuvant is any one
IFN-.alpha. or a CpG ODN. In some embodiments, the third adjuvant
is CpG 7909.
[0215] In some embodiments according to any one of the methods
described herein, the plurality of modified PBMCs is administered
prior to, concurrently with, or following administration of a
therapeutic agent. In some embodiments, the therapeutic agent
comprises one or more of an immune checkpoint inhibitor, a
chemotherapy, or a radiotherapy. In some embodiments, the
therapeutic agent comprises one or more cytokines.
[0216] Immune checkpoints are regulators of the immune system and
keep immune responses in check. Immune checkpoint inhibitors can be
employed to facilitate the enhancement of immune response. In some
embodiments, the composition comprising the plurality of modified
PBMCs is administered in combination with administration of an
immune checkpoint inhibitor. In some embodiments, the composition
comprising the plurality of modified PBMCs and the immune
checkpoint inhibitor are administered simultaneously. In some
embodiments, the composition comprising the plurality of modified
PBMCs and the immune checkpoint inhibitor are administered
sequentially.
[0217] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered prior to administration
of the immune checkpoint inhibitor. In some embodiments, the
composition comprising the plurality of modified PBMCs is
administered following administration of the immune checkpoint
inhibitor. For example, the composition comprising the plurality of
modified PBMCs is administered from about 1 hour to about 1 week
prior to administration of the immune checkpoint inhibitor. For
example, in some embodiments, the composition comprising the
plurality of modified PBMCs is administered about 1 hour, about 2
hours, about 3 hours, about 4 hours, about 6 hours, about 8 hours,
about 10 hours, about 12 hours, about 14 hours, about 16 hours,
about 18 hours, about 20 hours, about 24 hours, about 30 hours,
about 36 hours, about 42 hours, about 48 hours, about 60 hours,
about 3 days, about 4 days, about 5 days, about 6 days, or about 7
days prior to administration of the immune checkpoint inhibitor. In
some embodiments, the composition comprising the plurality of
modified PBMCs is administered from between about 1 hour and about
2 hours, from between about 2 hours and about 3 hours, from between
about 3 hours and about 4 hours, from between about 4 hours and
about 6 hours, from between about 6 hours and about 8 hours, from
between about 8 hours and about 10 hours, from between about 10
hours and about 12 hours, from between about 12 hours and about 14
hours, from between about 14 hours and about 16 hours, from between
about 16 hours and about 18 hours, from between about 18 hours and
about 20 hours, from between about 20 hours and about 24 hours,
from between about 24 hours and about 30 hours, from between about
30 hours and about 36 hours, from between about 36 hours and about
42 hours, from between about 42 hours and about 48 hours, from
between about 48 hours and about 60 hours, from between about 60
hours and about 3 days, from between about 3 days and about 4 days,
from between about 4 days and about 5 days, from between about 5
days and about 6 days, from between about 6 days and about 7 days
prior to administration of the immune checkpoint inhibitor.
[0218] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered about 7 days, about 10
days, about 14 days, about 18 days, about 21 days, about 24 days,
about 28 days, about 30 days, about 35 days, about 40 days, about
45 days, or about 50 days prior to administration of the immune
checkpoint inhibitor. In some embodiments, the composition
comprising the plurality of modified PBMCs is administered from
between about 7 days to about 10 days, from between about 10 days
and about 14 days, from between about 14 days and about 18 days,
from between about 18 days and about 21 days, from between about 21
days and about 24 days, from between about 24 days and about 28
days, from between about 28 days and about 30 days, from between
about 30 days and about 35 days, from between about 35 days and
about 40 days, from between about 40 days and about 45 days, or
from between about 45 days and about 50 days prior to
administration of the immune checkpoint inhibitor.
[0219] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered following
administration of the immune checkpoint inhibitor. For example, the
composition comprising the plurality of modified PBMCs is
administered from about 1 hour to about 1 week following
administration of the immune checkpoint inhibitor. For example, in
some embodiments, the composition comprising the plurality of
modified PBMCs is administered about 1 hour, about 2 hours, about 3
hours, about 4 hours, about 6 hours, about 8 hours, about 10 hours,
about 12 hours, about 14 hours, about 16 hours, about 18 hours,
about 20 hours, about 24 hours, about 30 hours, about 36 hours,
about 42 hours, about 48 hours, about 60 hours, about 3 days, about
4 days, about 5 days, about 6 days, or about 7 days following
administration of the immune checkpoint inhibitor. In some
embodiments, the composition comprising the plurality of modified
PBMCs is administered from between about 1 hour and about 2 hours,
from between about 2 hours and about 3 hours, from between about 3
hours and about 4 hours, from between about 4 hours and about 6
hours, from between about 6 hours and about 8 hours, from between
about 8 hours and about 10 hours, from between about 10 hours and
about 12 hours, from between about 12 hours and about 14 hours,
from between about 14 hours and about 16 hours, from between about
16 hours and about 18 hours, from between about 18 hours and about
20 hours, from between about 20 hours and about 24 hours, from
between about 24 hours and about 30 hours, from between about 30
hours and about 36 hours, from between about 36 hours and about 42
hours, from between about 42 hours and about 48 hours, from between
about 48 hours and about 60 hours, from between about 60 hours and
about 3 days, from between about 3 days and about 4 days, from
between about 4 days and about 5 days, from between about 5 days
and about 6 days, from between about 6 days and about 7 days
following administration of the immune checkpoint inhibitor.
[0220] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered about 7 days, about 10
days, about 14 days, about 18 days, about 21 days, about 24 days,
about 28 days, about 30 days, about 35 days, about 40 days, about
45 days, or about 50 days following administration of the immune
checkpoint inhibitor. In some embodiments, the composition
comprising the plurality of modified PBMCs is administered from
between about 7 days to about 10 days, from between about 10 days
and about 14 days, from between about 14 days and about 18 days,
from between about 18 days and about 21 days, from between about 21
days and about 24 days, from between about 24 days and about 28
days, from between about 28 days and about 30 days, from between
about 30 days and about 35 days, from between about 35 days and
about 40 days, from between about 40 days and about 45 days, or
from between about 45 days and about 50 days following
administration of the immune checkpoint inhibitor.
[0221] In some embodiments, the method comprises multiple
administration of the composition comprising the plurality of
modified PBMCs and/or multiple administration of the immune
checkpoint inhibitor. For example, in some embodiments, the method
comprises two administrations, three administrations, four
administrations, five administrations, six administrations, seven
administrations, eight administrations, nine administrations, ten
administrations, eleven administrations, twelve administrations,
thirteen administrations, fourteen administrations, or fifteen
administrations of the composition comprising the plurality of
modified PBMCs and/or the immune checkpoint inhibitor. For example,
in some embodiments, the method comprises less than five
administrations, less than ten administrations, less than fifteen
administrations, less than twenty administrations, less than
twenty-five administrations, less than thirty administrations, less
than fifty administrations, less than seventy-five administrations,
less than one hundred, or less than two hundred administrations of
the composition comprising the plurality of modified PBMCs and/or
the immune checkpoint inhibitor.
[0222] Exemplary immune checkpoint inhibitor is targeted to,
without limitation, PD-1, PD-L1, CTLA-4, LAG3, TIM-3, TIGIT, VISTA,
TIM1, B7-H4 (VTCN1) or BTLA. In some embodiments, the immune
checkpoint inhibitor is targeted to one or more of PD-1, PD-L1,
CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1) or BTLA. In
some embodiments, the immune checkpoint inhibitor is one or more
of: an antibody that binds to PD-1, an antibody that binds PD-L1,
an antibody that binds CTLA-4, an antibody that binds LAG3, or an
antibody that binds TIM-3, an antibody that binds TIGIT, an
antibody that binds VISTA, an antibody that binds TIM-1, an
antibody that binds B7-H4, or an antibody that binds BTLA. In
further embodiments, the antibody can be a full length antibody or
any variants, for example but not limited to, an antibody fragment,
a single chain variable fragment (ScFv), or a fragment
antigen-binding (Fab). In further embodiments, the antibody can be
bispecific, trispecific or multispecific. In some embodiments, the
immune checkpoint inhibitor is one or more chemical compounds that
binds to and/or inhibits one or more of PD-1, PD-L1, CTLA-4, LAG3,
TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1) or BTLA. In some
embodiments, the immune checkpoint inhibitor is one or more
peptides that binds to and/or inhibits one or more of PD-1, PD-L1,
CTLA-4, LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1) or BTLA. In
some embodiments, the immune checkpoint inhibitor is targeted to
PD-1. In some embodiments, the immune checkpoint inhibitor is
targeted to PD-L1.
[0223] Cytokines can be used in combination with any one of the
pluralities of modified PBMCs described herein to achieve additive
or synergistic effects against cancers, for example, HPV-associated
cancers. In some embodiments, the composition comprising the
plurality of modified PBMCs is administered in combination with
administration of one or more cytokines. In some embodiments, the
composition comprising the plurality of modified PBMCs and the
cytokine are administered simultaneously. In some embodiments, the
composition comprising the plurality of modified PBMCs and the
cytokine are administered sequentially.
[0224] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered prior to administration
of the cytokine. In some embodiments, the composition comprising
the plurality of modified PBMCs is administered following
administration of the cytokine. For example, the composition
comprising the plurality of modified PBMCs is administered from
about 1 hour to about 1 week prior to administration of the
cytokine. For example, in some embodiments, the composition
comprising the plurality of modified PBMCs is administered about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours,
about 8 hours, about 10 hours, about 12 hours, about 14 hours,
about 16 hours, about 18 hours, about 20 hours, about 24 hours,
about 30 hours, about 36 hours, about 42 hours, about 48 hours,
about 60 hours, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days prior to administration of the cytokine. In
some embodiments, the composition comprising the plurality of
modified PBMCs is administered from between about 1 hour and about
2 hours, from between about 2 hours and about 3 hours, from between
about 3 hours and about 4 hours, from between about 4 hours and
about 6 hours, from between about 6 hours and about 8 hours, from
between about 8 hours and about 10 hours, from between about 10
hours and about 12 hours, from between about 12 hours and about 14
hours, from between about 14 hours and about 16 hours, from between
about 16 hours and about 18 hours, from between about 18 hours and
about 20 hours, from between about 20 hours and about 24 hours,
from between about 24 hours and about 30 hours, from between about
30 hours and about 36 hours, from between about 36 hours and about
42 hours, from between about 42 hours and about 48 hours, from
between about 48 hours and about 60 hours, from between about 60
hours and about 3 days, from between about 3 days and about 4 days,
from between about 4 days and about 5 days, from between about 5
days and about 6 days, from between about 6 days and about 7 days
prior to administration of the cytokine.
[0225] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered about 7 days, about 10
days, about 14 days, about 18 days, about 21 days, about 24 days,
about 28 days, about 30 days, about 35 days, about 40 days, about
45 days, or about 50 days prior to administration of the cytokine.
In some embodiments, the composition comprising the plurality of
modified PBMCs is administered from between about 7 days to about
10 days, from between about 10 days and about 14 days, from between
about 14 days and about 18 days, from between about 18 days and
about 21 days, from between about 21 days and about 24 days, from
between about 24 days and about 28 days, from between about 28 days
and about 30 days, from between about 30 days and about 35 days,
from between about 35 days and about 40 days, from between about 40
days and about 45 days, or from between about 45 days and about 50
days prior to administration of the cytokine.
[0226] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered following
administration of the cytokine. For example, the composition
comprising the plurality of modified PBMCs is administered from
about 1 hour to about 1 week following administration of the
cytokine. For example, in some embodiments, the composition
comprising the plurality of modified PBMCs is administered about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours,
about 8 hours, about 10 hours, about 12 hours, about 14 hours,
about 16 hours, about 18 hours, about 20 hours, about 24 hours,
about 30 hours, about 36 hours, about 42 hours, about 48 hours,
about 60 hours, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days following administration of the cytokine. In
some embodiments, the composition comprising the plurality of
modified PBMCs is administered from between about 1 hour and about
2 hours, from between about 2 hours and about 3 hours, from between
about 3 hours and about 4 hours, from between about 4 hours and
about 6 hours, from between about 6 hours and about 8 hours, from
between about 8 hours and about 10 hours, from between about 10
hours and about 12 hours, from between about 12 hours and about 14
hours, from between about 14 hours and about 16 hours, from between
about 16 hours and about 18 hours, from between about 18 hours and
about 20 hours, from between about 20 hours and about 24 hours,
from between about 24 hours and about 30 hours, from between about
30 hours and about 36 hours, from between about 36 hours and about
42 hours, from between about 42 hours and about 48 hours, from
between about 48 hours and about 60 hours, from between about 60
hours and about 3 days, from between about 3 days and about 4 days,
from between about 4 days and about 5 days, from between about 5
days and about 6 days, from between about 6 days and about 7 days
following administration of the cytokine.
[0227] Exemplary cytokines include but are not limited to
chemokines, interferons, interleukins, lymphokines, and tumour
necrosis factors. In some embodiments, the cytokine enhances
cellular immune responses. In some embodiments, the cytokine
enhances antibody responses. In some embodiments, the cytokine is a
type I cytokine. In some embodiments, the cytokine is a type 2
cytokine. In some embodiments, the cytokine comprises one or more
of: IL-2, IL-15, IL-10, IL-12, IFN-.alpha., or IL-21. In some
embodiments, the cytokine comprises IL-15.
[0228] Chemotherapy can be used in combination with any one of the
pluralities of modified PBMCs described herein to achieve additive
or synergistic effects against cancers, for example, HPV-associated
cancers. In some embodiments, the composition comprising the
plurality of modified PBMCs is administered in combination with
administration of a chemotherapy. In some embodiments, the
composition comprising the plurality of modified PBMCs and the
chemotherapy are administered simultaneously. In some embodiments,
the composition comprising the plurality of modified PBMCs and the
chemotherapy are administered sequentially.
[0229] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered prior to administration
of the chemotherapy. In some embodiments, the composition
comprising the plurality of modified PBMCs is administered
following administration of the chemotherapy. For example, the
composition comprising the plurality of modified PBMCs is
administered from about 1 hour to about 1 week prior to
administration of the chemotherapy. For example, in some
embodiments, the composition comprising the plurality of modified
PBMCs is administered about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 6 hours, about 8 hours, about 10 hours, about
12 hours, about 14 hours, about 16 hours, about 18 hours, about 20
hours, about 24 hours, about 30 hours, about 36 hours, about 42
hours, about 48 hours, about 60 hours, about 3 days, about 4 days,
about 5 days, about 6 days, or about 7 days prior to administration
of the chemotherapy. In some embodiments, the composition
comprising the plurality of modified PBMCs is administered from
between about 1 hour and about 2 hours, from between about 2 hours
and about 3 hours, from between about 3 hours and about 4 hours,
from between about 4 hours and about 6 hours, from between about 6
hours and about 8 hours, from between about 8 hours and about 10
hours, from between about 10 hours and about 12 hours, from between
about 12 hours and about 14 hours, from between about 14 hours and
about 16 hours, from between about 16 hours and about 18 hours,
from between about 18 hours and about 20 hours, from between about
20 hours and about 24 hours, from between about 24 hours and about
30 hours, from between about 30 hours and about 36 hours, from
between about 36 hours and about 42 hours, from between about 42
hours and about 48 hours, from between about 48 hours and about 60
hours, from between about 60 hours and about 3 days, from between
about 3 days and about 4 days, from between about 4 days and about
5 days, from between about 5 days and about 6 days, from between
about 6 days and about 7 days prior to administration of the
chemotherapy.
[0230] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered following
administration of the chemotherapy. For example, the composition
comprising the plurality of modified PBMCs is administered from
about 1 hour to about 1 week following administration of the
chemotherapy. For example, in some embodiments, the composition
comprising the plurality of modified PBMCs is administered about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours,
about 8 hours, about 10 hours, about 12 hours, about 14 hours,
about 16 hours, about 18 hours, about 20 hours, about 24 hours,
about 30 hours, about 36 hours, about 42 hours, about 48 hours,
about 60 hours, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days following administration of the chemotherapy.
In some embodiments, the composition comprising the plurality of
modified PBMCs is administered from between about 1 hour and about
2 hours, from between about 2 hours and about 3 hours, from between
about 3 hours and about 4 hours, from between about 4 hours and
about 6 hours, from between about 6 hours and about 8 hours, from
between about 8 hours and about 10 hours, from between about 10
hours and about 12 hours, from between about 12 hours and about 14
hours, from between about 14 hours and about 16 hours, from between
about 16 hours and about 18 hours, from between about 18 hours and
about 20 hours, from between about 20 hours and about 24 hours,
from between about 24 hours and about 30 hours, from between about
30 hours and about 36 hours, from between about 36 hours and about
42 hours, from between about 42 hours and about 48 hours, from
between about 48 hours and about 60 hours, from between about 60
hours and about 3 days, from between about 3 days and about 4 days,
from between about 4 days and about 5 days, from between about 5
days and about 6 days, from between about 6 days and about 7 days
following administration of the chemotherapy.
[0231] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered about 7 days, about 10
days, about 14 days, about 18 days, about 21 days, about 24 days,
about 28 days, about 30 days, about 35 days, about 40 days, about
45 days, or about 50 days following administration of the
chemotherapy. In some embodiments, the composition comprising the
plurality of modified PBMCs is administered from between about 7
days to about 10 days, from between about 10 days and about 14
days, from between about 14 days and about 18 days, from between
about 18 days and about 21 days, from between about 21 days and
about 24 days, from between about 24 days and about 28 days, from
between about 28 days and about 30 days, from between about 30 days
and about 35 days, from between about 35 days and about 40 days,
from between about 40 days and about 45 days, or from between about
45 days and about 50 days following administration of the
chemotherapy.
[0232] In some embodiments, the method comprises multiple
administration of the composition comprising the plurality of
modified PBMCs and/or multiple administration of the chemotherapy.
For example, in some embodiments, the method comprises two
administrations, three administrations, four administrations, five
administrations, six administrations, seven administrations, eight
administrations, nine administrations, ten administrations, eleven
administrations, twelve administrations, thirteen administrations,
fourteen administrations, or fifteen administrations of the
composition comprising the plurality of modified PBMCs and/or the
chemotherapy. For example, in some embodiments, the method
comprises less than five administrations, less than ten
administrations, less than fifteen administrations, less than
twenty administrations, less than twenty-five administrations, less
than thirty administrations, less than fifty administrations, less
than seventy-five administrations, less than one hundred, or less
than two hundred administrations of the composition comprising the
plurality of modified PBMCs and/or the chemotherapy.
[0233] Exemplary chemotherapy can be cell cycle dependent or cell
cycle independent. In some embodiments, the chemotherapy comprises
one or more chemotherapeutic agents. In some embodiments, a
chemotherapeutic agent can target one or more of cell division,
DNA, or metabolism in cancer. In some embodiments, the
chemotherapeutic agent is a platinum-based agent, such as but not
limited to cisplatin, oxaliplatin or carboplatin. In some
embodiments, the chemotherapeutic agent is a taxane (such as
docetaxel or paclitaxel). In some embodiments, the chemotherapeutic
agent is 5-fluorouracil, doxorubicin, or irinotecan. In some
embodiments, the chemotherapeutic agent is one or more of: an
alkylating agent, an antimetabolite, an antitumor antibiotic, a
topoisomerase inhibitor or a mitotic inhibitor. In some
embodiments, the chemotherapy comprises cisplatin.
[0234] Radiotherapy can be used in combination with any one of the
pluralities of modified PBMCs described herein to achieve additive
or synergistic effects against cancers, for example, HPV-associated
cancers. In some embodiments, the composition comprising the
plurality of modified PBMCs is administered in combination with
administration of a radiotherapy. In some embodiments, the
composition comprising the plurality of modified PBMCs and the
radiotherapy are administered simultaneously. In some embodiments,
the composition comprising the plurality of modified PBMCs and the
radiotherapy are administered sequentially. In some embodiments,
the composition comprising the plurality of modified PBMCs is
administered in combination with administration of a radiotherapy,
in combination with a chemotherapy, and/or in combination with an
immune checkpoint inhibitor.
[0235] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered prior to administration
of the radiotherapy. In some embodiments, the composition
comprising the plurality of modified PBMCs is administered
following administration of the radiotherapy. For example, the
composition comprising the plurality of modified PBMCs is
administered from about 1 hour to about 1 week prior to
administration of the radiotherapy. For example, in some
embodiments, the composition comprising the plurality of modified
PBMCs is administered about 1 hour, about 2 hours, about 3 hours,
about 4 hours, about 6 hours, about 8 hours, about 10 hours, about
12 hours, about 14 hours, about 16 hours, about 18 hours, about 20
hours, about 24 hours, about 30 hours, about 36 hours, about 42
hours, about 48 hours, about 60 hours, about 3 days, about 4 days,
about 5 days, about 6 days, or about 7 days prior to administration
of the radiotherapy. In some embodiments, the composition
comprising the plurality of modified PBMCs is administered from
between about 1 hour and about 2 hours, from between about 2 hours
and about 3 hours, from between about 3 hours and about 4 hours,
from between about 4 hours and about 6 hours, from between about 6
hours and about 8 hours, from between about 8 hours and about 10
hours, from between about 10 hours and about 12 hours, from between
about 12 hours and about 14 hours, from between about 14 hours and
about 16 hours, from between about 16 hours and about 18 hours,
from between about 18 hours and about 20 hours, from between about
20 hours and about 24 hours, from between about 24 hours and about
30 hours, from between about 30 hours and about 36 hours, from
between about 36 hours and about 42 hours, from between about 42
hours and about 48 hours, from between about 48 hours and about 60
hours, from between about 60 hours and about 3 days, from between
about 3 days and about 4 days, from between about 4 days and about
5 days, from between about 5 days and about 6 days, from between
about 6 days and about 7 days prior to administration of the
radiotherapy.
[0236] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered following
administration of the radiotherapy. For example, the composition
comprising the plurality of modified PBMCs is administered from
about 1 hour to about 1 week following administration of the
radiotherapy. For example, in some embodiments, the composition
comprising the plurality of modified PBMCs is administered about 1
hour, about 2 hours, about 3 hours, about 4 hours, about 6 hours,
about 8 hours, about 10 hours, about 12 hours, about 14 hours,
about 16 hours, about 18 hours, about 20 hours, about 24 hours,
about 30 hours, about 36 hours, about 42 hours, about 48 hours,
about 60 hours, about 3 days, about 4 days, about 5 days, about 6
days, or about 7 days following administration of the radiotherapy.
In some embodiments, the composition comprising the plurality of
modified PBMCs is administered from between about 1 hour and about
2 hours, from between about 2 hours and about 3 hours, from between
about 3 hours and about 4 hours, from between about 4 hours and
about 6 hours, from between about 6 hours and about 8 hours, from
between about 8 hours and about 10 hours, from between about 10
hours and about 12 hours, from between about 12 hours and about 14
hours, from between about 14 hours and about 16 hours, from between
about 16 hours and about 18 hours, from between about 18 hours and
about 20 hours, from between about 20 hours and about 24 hours,
from between about 24 hours and about 30 hours, from between about
30 hours and about 36 hours, from between about 36 hours and about
42 hours, from between about 42 hours and about 48 hours, from
between about 48 hours and about 60 hours, from between about 60
hours and about 3 days, from between about 3 days and about 4 days,
from between about 4 days and about 5 days, from between about 5
days and about 6 days, from between about 6 days and about 7 days
following administration of the radiotherapy.
[0237] In some embodiments, the composition comprising the
plurality of modified PBMCs is administered about 7 days, about 10
days, about 14 days, about 18 days, about 21 days, about 24 days,
about 28 days, about 30 days, about 35 days, about 40 days, about
45 days, or about 50 days following administration of the
radiotherapy. In some embodiments, the composition comprising the
plurality of modified PBMCs is administered from between about 7
days to about 10 days, from between about 10 days and about 14
days, from between about 14 days and about 18 days, from between
about 18 days and about 21 days, from between about 21 days and
about 24 days, from between about 24 days and about 28 days, from
between about 28 days and about 30 days, from between about 30 days
and about 35 days, from between about 35 days and about 40 days,
from between about 40 days and about 45 days, or from between about
45 days and about 50 days following administration of the
radiotherapy.
[0238] In some embodiments, the method comprises multiple
administration of the composition comprising the plurality of
modified PBMCs and/or multiple administration of the radiotherapy.
For example, in some embodiments, the method comprises two
administrations, three administrations, four administrations, five
administrations, six administrations, seven administrations, eight
administrations, nine administrations, ten administrations, eleven
administrations, twelve administrations, thirteen administrations,
fourteen administrations, or fifteen administrations of the
composition comprising the plurality of modified PBMCs and/or the
radiotherapy. For example, in some embodiments, the method
comprises less than five administrations, less than ten
administrations, less than fifteen administrations, less than
twenty administrations, less than twenty-five administrations, less
than thirty administrations, less than fifty administrations, less
than seventy-five administrations, less than one hundred, or less
than two hundred administrations of the composition comprising the
plurality of modified PBMCs and/or the radiotherapy.
[0239] In some embodiments, there is provided a plurality of PBMCs
comprising an antigen for use in a method of stimulating an immune
response in an individual according to any one of the methods
described herein.
[0240] In some methods according to any one of the methods
described herein, the method stimulates an immune response against
an HPV antigen in an individual. Papillomaviruses are small
nonenveloped DNA viruses with a virion size of .about.55 nm in
diameter. More than 100 HPV genotypes are completely characterized,
and a higher number is presumed to exist. HPV is a known cause of
cervical cancers, as well as some vulvar, vaginal, penile,
oropharyngeal, anal, and rectal cancers. Although most HPV
infections are asymptomatic and clear spontaneously, persistent
infections with one of the oncogenic HPV types can progress to
precancer or cancer. Other HPV-associated diseases can include
common warts, plantar warts, flat warts, anogenital warts, anal
lesions, epidermodysplasia, focal epithelial hyperplasia, mouth
papillomas, verrucous cysts, laryngeal papillomatosis, squamous
intraepithelial lesions (SILs), cervical intraepithelial neoplasia
(CIN), vulvar intraepithelial neoplasia (VIN) and vaginal
intraepithelial neoplasia (VAIN). Many of the known human
papillomavirus (HPV) types cause benign lesions with a subset being
oncogenic. Based on epidemiologic and phylogenetic relationships,
HPV types are classified into fifteen "high risk types" (HPV 16,
18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and
three "probable high risk types" (HPV 26, 53, and 66), which
together are known to manifest as low and high grade cervical
changes and cancers, as well as other anogential cancers such as
vulval, vaginal, penile, anal, and perianal cancer, as well as head
and neck cancers. Recently, the association of high risk types HPV
16 and 18 with breast cancer was also described. Eleven HPV types
classified as "low risk types" (HPV 6, 11, 40, 42, 43, 44, 54, 61,
70, 72, and 81) are known to manifest as benign low-grade cervical
changes, genital warts and recurrent respiratory papillomatosis.
Cutaneous HPV types 5, 8, and 92 are associated with skin cancer.
In some HPV-associated cancers, the immune system is depressed and
correspondingly, the antitumor response is significantly impaired.
See Suresh and Burtness, Am J Hematol Oncol 13(6):20-27 (2017).
[0241] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs. In some embodiments, the plurality of
PBMCs comprises a nucleic acid encoding an antigen. In some
embodiments, the plurality of PBMCs comprises an mRNA encoding an
antigen.
[0242] PBMC Compositions
[0243] As used herein, PBMCs may be isolated by leukapheresis from
whole blood obtained from an individual. Also provided are PBMC
compositions are reconstituted by mixing different pools of PBMCs
from the same individual or different individuals. In other
examples, PBMCs may also be reconstituted by mixing different
populations of cells into a mixed cell composition with a generated
profile. In some embodiments, the populations of cells used for
reconstituting PBMCs are mixed populations of cells (such as a
mixture of one or more of T cells, B cells, NK cells or monocytes).
In some embodiments, the populations of cells used for
reconstituting PBMCs are purified populations of cells (such as
purified T cells, B cells, NK cells or monocytes). In additional
examples, the different populations of cells used in reconstituting
a PBMC composition can be isolated from the same individual (e.g.
autologous) or isolated from different individuals (e.g. allogenic
and/or heterologous).
[0244] Therefore in some embodiments according to any one of the
methods, compositions or pluralities of modified PBMCs described
herein, the plurality of input PBMCs comprises one or more of T
cells, B cells, NK cells, monocytes, dendritic cells or NK-T cells.
In some embodiments, the plurality of input PBMCs comprises T
cells, B cells, NK cells, monocytes, dendritic cells or NK-T cells.
In some embodiments, the plurality of input PBMCs comprises one or
more of CD3+ T cells, CD20+ B cells, CD14+ monocytes, CD56+NK
cells. In some embodiments, the plurality of input PBMCs comprises
T cells, B cells, NK cells and monocytes, and the ratio of T cells,
B cells, NK cells and monocytes to the total number of PBMCs in the
plurality of input PBMCs is essentially the same as the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in whole blood. In some embodiments, the plurality of input PBMCs
comprises T cells, B cells, NK cells and monocytes, and the ratio
of T cells, B cells, NK cells and monocytes to the total number of
PBMCs in the plurality of input PBMCs is essentially the same as
the ratio of T cells, B cells, NK cells and monocytes to the total
number of PBMCs in a leukapheresis product from whole blood. In
some embodiments, the plurality of input PBMCs comprises T cells, B
cells, NK cells and monocytes, and the ratio of T cells, B cells,
NK cells and monocytes to the total number of PBMCs in the
plurality of input PBMCs differs by not more than any one of 1%,
2%, 5%, 10% 15%, 20%, 25%, 30%, 40%, or 50% from the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in whole blood. In some embodiments, the plurality of input PBMCs
comprises T cells, B cells, NK cells and monocytes, and the ratio
of T cells, B cells, NK cells and monocytes to the total number of
PBMCs in the plurality of input PBMCs differs by not more than any
one of 10% from the ratio of T cells, B cells, NK cells and
monocytes to the total number of PBMCs in whole blood. In some
embodiments, the plurality of input PBMCs comprises T cells, B
cells, NK cells and monocytes, and the ratio of T cells, B cells,
NK cells and monocytes to the total number of PBMCs in the
plurality of input PBMCs differs by not more than any one of 1%,
2%, 5%, 10% 15%, 20%, 25%, 30%, 40%, or 50% from the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in a leukapheresis product from whole blood. In some embodiments,
the plurality of input PBMCs comprises T cells, B cells, NK cells
and monocytes, and the ratio of T cells, B cells, NK cells and
monocytes to the total number of PBMCs in the plurality of input
PBMCs differs by not more than any one of 10% from the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in a leukapheresis product from whole blood.
[0245] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein,
about 25% to about 70% of the modified PBMCs are T cells. In some
embodiments, about 2.5% to about 14% of the modified PBMCs are B
cells. In some embodiments, about 3.5% to about 35% of the modified
PBMCs are NK cells. In some embodiments, about 4% to about 25% of
the modified PBMCs are NK cells.
[0246] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, at
least about 90% to about 99% of the input PBMCs consist of T cells,
B cells, NK cells and monocytes. In some embodiments, at least any
one of about 80% to about 85%, about 85% to about 90%, about 90% to
about 95% or about 95% to about 99% of the input PBMCs consist of T
cells, B cells, NK cells and monocytes. In some embodiments, at
least about any one of 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the
input PBMCs consist of T cells, B cells, NK cells and monocytes. In
some embodiments, at least about 90% of the input PBMCs consist of
T cells, B cells, NK cells and monocytes. In some embodiments, the
input PBMCs consist of T cells, B cells, NK cells and
monocytes.
[0247] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, at
least about 90% to about 99% of the modified PBMCs consist of T
cells, B cells, NK cells and monocytes. In some embodiments, at
least any one of about 80% to about 85%, about 85% to about 90%,
about 90% to about 95% or about 95% to about 99% of the modified
PBMCs consist of T cells, B cells, NK cells and monocytes. In some
embodiments, at least about any one of 80%, 81%, 82%, 83%, 84%,
85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,
98%, or 99% of the modified PBMCs consist of T cells, B cells, NK
cells and monocytes. In some embodiments, at least about 90% of the
modified PBMCs consist of T cells, B cells, NK cells and monocytes.
In some embodiments, the modified PBMCs consist of T cells, B
cells, NK cells and monocytes.
[0248] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, at
least about any one of 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%,
60%, 65%, 70%, or 75% of the input PBMCs are T cells. In some
embodiments, at least about 25% of the input PBMCs are T cells. In
some embodiments, at least about any one of 0.5%, 1%, 1.5%, 2%,
2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%,
15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the input PBMCs are B
cells. In some embodiments, at least about 2.5% of the input PBMCs
are B cells. In some embodiments, at least about any one of 0.5%,
1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%,
12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30% of the
input PBMCs are NK cells. In some embodiments, at least about 3.5%
of the input PBMCs are NK cells. In some embodiments, at least
about any one of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%,
16%, 18%, 20%, 25%, 30%, 35% or 40% of the input PBMCs are
monocytes. In some embodiments, at least about 4% of the input
PBMCs are monocytes. In some embodiments, at least about 25% of the
input PBMCs are T cells; at least about 2.5% of the input PBMCs are
B cells; at least about 3.5% of the input PBMCs are NK cells; and
at least about 4% of the input PBMCs are monocytes.
[0249] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, at
least about any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 65%, or 70% of the modified PBMCs are T cells. In some
embodiments, at least about 20% of the modified PBMCs are T cells.
In some embodiments, at least about any one of 0.25%, 0.5%, 1%,
1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%,
13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25% or 30% of the modified
PBMCs are B cells. In some embodiments, at least about 2% of the
modified PBMCs are B cells. In some embodiments, at least about any
one of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%,
10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, or 30%
of the modified PBMCs are NK cells. In some embodiments, at least
about 3% of the modified PBMCs are NK cells. In some embodiments,
at least about any one of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%,
12%, 14%, 16%, 18%, 20%, 25%, 30%, 35% or 40% of the modified PBMCs
are monocytes. In some embodiments, at least about 3% of the
modified PBMCs are monocytes. In some embodiments, at least about
20% of the modified PBMCs are T cells; at least about 2% of the
modified PBMCs are B cells; at least about 3% of the modified PBMCs
are NK cells; and at least about 3% of the modified PBMCs are
monocytes.
[0250] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, not
more than about any one of 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%,
80%, 85%, or 90% of the input PBMCs are T cells. In some
embodiments, not more than about 70% of the input PBMCs are T
cells. In some embodiments, not more than about any one of 5%, 10%,
12%, 14%, 16%, 18%, 20%, 22%, 25%, 30%, 35%, 40%, or 50% of the
input PBMCs are B cells. In some embodiments, not more than about
14% of the input PBMCs are B cells. In some embodiments, not more
than about any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%
or 60% of the input PBMCs are NK cells. In some embodiments, not
more than about 35% of the input PBMCs are NK cells. In some
embodiments, not more than about any one of 5%, 10%, 12%, 14%, 16%,
18%, 20%, 22%, 25%, 30%, 35%, 40%, or 50% of the input PBMCs are
monocytes. In some embodiments, not more than about 4% of the input
PBMCs are monocytes. In some embodiments, not more than about 25%
of the input PBMCs are T cells; not more than about 2.5% of the
input PBMCs are B cells; not more than about 3.5% of the input
PBMCs are NK cells; and not more than about 4% of the input PBMCs
are monocytes.
[0251] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, not
more than about any one of 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,
50%, 55%, 60%, 65%, or 70% of the modified PBMCs are T cells. In
some embodiments, not more than about 20% of the modified PBMCs are
T cells. In some embodiments, not more than about any one of 0.25%,
0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%, 5%, 6%, 7%, 7.5%, 8%, 9%, 10%,
11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25% or 30% of the
modified PBMCs are B cells. In some embodiments, not more than
about 2% of the modified PBMCs are B cells. In some embodiments,
not more than about any one of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 4%,
5%, 6%, 7%, 7.5%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%,
18%, 19%, 20%, 25%, or 30% of the modified PBMCs are NK cells. In
some embodiments, not more than about 3% of the modified PBMCs are
NK cells. In some embodiments, not more than about any one of 1%,
2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 12%, 14%, 16%, 18%, 20%, 25%,
30%, 35% or 40% of the modified PBMCs are monocytes. In some
embodiments, not more than about 3% of the modified PBMCs are
monocytes. In some embodiments, not more than about 20% of the
modified PBMCs are T cells; not more than about 2% of the modified
PBMCs are B cells; not more than about 3% of the modified PBMCs are
NK cells; and not more than about 3% of the modified PBMCs are
monocytes.
[0252] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein,
about any one of 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%,
40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to
70%, or 70% to 75% of the modified PBMCs are T cells. In some
embodiments, about 25% to about 70% of the modified PBMCs are T
cells. In some embodiments, about any one of 1% to 2.5%, 2.5% to
4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to
16%, 16% to 20% or 20% to 25% of the modified PBMCs are B cells. In
some embodiments, about 2.5% to about 14% of the modified PBMCs are
B cells. In some embodiments, about any one of 1% to 2%, 2% to
3.5%, 3.5% to 5%, 5% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14%
to 16%, 16% to 20% or 20% to 25% of the modified PBMCs are B cells.
In some embodiments, about 3.5% to about 35% of the modified PBMCs
are NK cells. In some embodiments, about any one of 2% to 4%, 4% to
6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%, 16% to
20%, 20% to 25%, 25% to 30%, 30% to 35%, or 35% to 40% of the
modified PBMCs are monocytes. In some embodiments, about 4% to
about 25% of the modified PBMCs are monocytes. In some embodiments,
about 25% to about 70% of the modified PBMCs are T cells, about
2.5% to about 14% of the modified PBMCs are B cells, about 3.5% to
about 35% of the modified PBMCs are NK cells, and about 4% to about
25% of the modified PBMCs are NK cells.
[0253] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein,
about any one of 20% to 25%, 25% to 30%, 30% to 35%, 35% to 40%,
40% to 45%, 45% to 50%, 50% to 55%, 55% to 60%, 60% to 65%, 65% to
70%, or 70% to 75% of the modified PBMCs are T cells. In some
embodiments, about 25% to about 70% of the modified PBMCs are T
cells. In some embodiments, about any one of 1% to 2.5%, 2.5% to
4%, 4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to
16%, 16% to 20% or 20% to 25% of the modified PBMCs are B cells. In
some embodiments, about 2.5% to about 14% of the modified PBMCs are
B cells. In some embodiments, about any one of 1% to 2%, 2% to
3.5%, 3.5% to 5%, 5% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14%
to 16%, 16% to 20% or 20% to 25% of the modified PBMCs are NK
cells. In some embodiments, about 3.5% to about 35% of the modified
PBMCs are NK cells. In some embodiments, about any one of 2% to 4%,
4% to 6%, 6% to 8%, 8% to 10%, 10% to 12%, 12% to 14%, 14% to 16%,
16% to 20%, 20% to 25%, 25% to 30%, 30% to 35%, or 35% to 40% of
the modified PBMCs are monocytes. In some embodiments, about 4% to
about 25% of the modified PBMCs are monocytes. In some embodiments,
about 25% to about 70% of the modified PBMCs are T cells, about
2.5% to about 14% of the modified PBMCs are B cells, about 3.5% to
about 35% of the modified PBMCs are NK cells, and about 4% to about
25% of the modified PBMCs are NK cells.
[0254] As used herein, PBMCs can also be generated after
manipulating the composition of a mixed cell population of
mononuclear blood cells (such as lymphocytes and monocytes). In
some instances, the input PBMCs are generated after reducing (such
as depleting) certain subpopulations (such as B cells) within a
mixed cell population of mononuclear blood cells. The composition
in a mixed cell population of mononuclear blood cells in an
individual can be manipulated to make the cell population more
closely resemble a leukapheresis product from whole blood in the
same individual. In other examples, the composition in a mixed cell
population of mononuclear blood cells (for example, mouse
splenocytes) can also be manipulated to make the cell population
more closely resemble human PBMCs isolated from a leukapheresis
product from human whole blood.
[0255] In some embodiments, the construction-mediated delivery does
not differentially modulate the viability of different
subpopulations (such as B cells, T cells, NK cells or monocytes)
within PBMCs in a significant manner. In some embodiments, the
conditioning process does not differentially modulate the viability
of different subpopulations within PBMCs in a significant manner.
In some embodiments, the further addition of agents (including but
not limited to any one of: biopreservation agents or agents that
enhance the function and/or viability of PBMCs) does not
differentially modulate the viability of various subpopulations
within PBMCs in a significant manner. Therefore in some embodiments
according to any one of the methods, compositions or pluralities of
modified PBMCs described herein, the percentage of T cells within
the plurality of modified PBMCs and the percentage of T cells
within the plurality of input PBMCs differ by no more than about
10% by number. In some embodiments, the percentage of T cells
within the plurality of modified PBMCs and the percentage of T
cells within the plurality of input PBMCs differ by no more than
about any one of 5%, 8%, 10%, 12%, 14%, 16%, 18% or 20% by number.
In some embodiments, the percentage of B cells within the plurality
of modified PBMCs and the percentage of B cells within the
plurality of input PBMCs differ by no more than about 10% by
number. In some embodiments, the percentage of B cells within the
plurality of modified PBMCs and the percentage of B cells within
the plurality of input PBMCs differ by no more than about any one
of 5%, 8%, 10%, 12%, 14%, 16%, 18% or 20% by number. In some
embodiments, the percentage of NK cells within the plurality of
modified PBMCs and the percentage of NK cells within the plurality
of input PBMCs differ by no more than about 10% by number. In some
embodiments, the percentage of NK cells within the plurality of
modified PBMCs and the percentage of NK cells within the plurality
of input PBMCs differ by no more than about any one of 5%, 8%, 10%,
12%, 14%, 16%, 18% or 20% by number. In some embodiments, the
percentage of monocytes within the plurality of modified PBMCs and
the percentage of monocytes within the plurality of input PBMCs
differ by no more than about 10% by number. In some embodiments,
the percentage of monocytes within the plurality of modified PBMCs
and the percentage of monocytes within the plurality of input PBMCs
differ by no more than about any one of 5%, 8%, 10%, 12%, 14%, 16%,
18% or 20% by number.
Antigens
[0256] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen is a disease-associated antigen. In some embodiments, the
antigen is derived from peptides or mRNA isolated from a diseased
cell. In some embodiments, the antigen is a non-self antigen. In
some embodiments, the antigen is a tumor antigen, viral antigen,
bacterial antigen, or fungal antigen. In some embodiments, the
antigen is derived from a lysate, such as a lysate of disease
cells. In some embodiments, the antigen is derived from a tumor
lysate. In some embodiments, the antigen is a tumor antigen or a
tumor associated antigen. In some embodiments, the antigen is
associated with a cancer. In some embodiments, the cancer is head
and neck cancer, cervical cancer, vulvar cancer, vaginal cancer,
penile cancer, anal cancer, perianal cancer, anogenital cancer,
oral cancer or salivary cancer. In some embodiments, the antigen is
a head and neck cancer antigen, a cervical cancer antigen, a vulvar
cancer antigen, a vaginal cancer antigen, a penile cancer antigen,
an anal cancer antigen, a perianal cancer antigen, an anogenital
cancer antigen, an oral cancer antigen, a salivary cancer antigen,
a breast cancer antigen, a skin cancer antigen, a bladder cancer
antigen, a colon cancer, a rectal cancer antigen, an endometrial
cancer antigen, a kidney cancer antigen, a leukemia antigen, a lung
cancer antigen, a melanoma antigen, a non-Hodgkin lymphoma antigen,
a pancreatic cancer antigen, a prostate cancer antigen, or a
thyroid cancer antigen, In some embodiments, the cancer is a solid
cancer. In some embodiments, the cancer is a hematologic cancer. In
some embodiments, the cancer is a virus-associated cancer. In some
embodiments, the cancer is a HPV-associated cancer. In some
embodiments, the cancer is a localized cancer. In some embodiments,
the cancer is a metastatic cancer. In some embodiments, the antigen
is associated with an infectious disease. In some embodiments, the
infectious disease is associated with HIV, HPV, EBV, MCV, HBV or
HCV.
[0257] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs.
[0258] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen is a human papillomavirus (HPV) antigen. Papillomaviruses
are small nonenveloped DNA viruses with a virion size of .about.55
nm in diameter. More than 100 HPV genotypes are completely
characterized, and a higher number is presumed to exist. HPV is a
known cause of cervical cancers, as well as some vulvar, vaginal,
penile, oropharyngeal, anal, and rectal cancers. Although most HPV
infections are asymptomatic and clear spontaneously, persistent
infections with one of the oncogenic HPV types can progress to
precancer or cancer. Other HPV-associated diseases can include
common warts, plantar warts, flat warts, anogenital warts, anal
lesions, epidermodysplasia, focal epithelial hyperplasia, mouth
papillomas, verrucous cysts, laryngeal papillomatosis, squamous
intraepithelial lesions (SILs), cervical intraepithelial neoplasia
(CIN), vulvar intraepithelial neoplasia (VIN) and vaginal
intraepithelial neoplasia (VAIN). Many of the known human
papillomavirus (HPV) types cause benign lesions with a subset being
oncogenic. Based on epidemiologic and phylogenetic relationships,
HPV types are classified into fifteen "high risk types" (HPV 16,
18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68, 73, and 82) and
three "probable high risk types" (HPV 26, 53, and 66), which
together are known to manifest as low and high grade cervical
changes and cancers, as well as other anogential cancers such as
vulval, vaginal, penile, anal, and perianal cancer, as well as head
and neck cancers. Recently, the association of high risk types HPV
16 and 18 with breast cancer was also described. Eleven HPV types
classified as "low risk types" (HPV 6, 11, 40, 42, 43, 44, 54, 61,
70, 72, and 81) are known to manifest as benign low-grade cervical
changes, genital warts and recurrent respiratory papillomatosis.
Cutaneous HPV types 5, 8, and 92 are associated with skin cancer.
In some HPV-associated cancers, the immune system is depressed and
correspondingly, the antitumor response is significantly impaired.
See Suresh and Burtness, Am J Hematol Oncol 13(6):20-27 (2017). In
some embodiments, the antigen is a pool of multiple polypeptides
that elicit a response against the same and or different antigens.
In some embodiments, an antigen in the pool of multiple antigens
does not decrease the immune response directed toward other
antigens in the pool of multiple antigens. In some embodiments, the
HPV antigen is a polypeptide comprising an antigenic HPV epitope
and one or more heterologous peptide sequences. In some
embodiments, the HPV antigen complexes with itself, with other
antigens, or with the adjuvant. In some embodiments, the HPV is
HPV-16 or HPV-18. In some embodiments, the HPV antigen is comprised
of an HLA-A2-specific epitope. In some embodiments, the HPV antigen
is an HPV E6 antigen or an HPV E7 antigen. In some embodiments, the
antigen comprises a peptide derived from HPV E6 and/or E7. In some
embodiments, the antigen comprises an HLA-A2-restricted peptide
derived from HPV E6 and/or E7. In some embodiments, the
HLA-A2-restricted peptide comprises the amino acid sequence of any
one of SEQ ID NOs: 1-4. In some embodiments, the HPV antigen
comprises an amino acid sequence with at least 90% similarity to
any one of SEQ ID NOs: 18-25. In some embodiments, the HPV antigen
comprises an amino acid sequence with at least 90% similarity to
SEQ ID NO: 19. In some embodiments, the HPV antigen comprises an
amino acid sequence with at least 90% similarity to SEQ ID NO: 23.
In some embodiments, the HPV antigen comprises the amino acid
sequence of SEQ ID NO: 19. In a preferred embodiment, the HPV
antigen consists of the amino acid sequence of SEQ ID NO: 19. In
some embodiments, the HPV antigen comprises the amino acid sequence
of SEQ ID NO: 23. In a preferred embodiment, the HPV antigen
consists of the amino acid sequence of SEQ ID NO: 23. In some
embodiments, the antigen comprises the amino acid sequence of any
one of SEQ ID NOs: 18-25. In some embodiments, the antigen is a
plurality of antigens comprising at least one of the amino acid
sequences of any one of SEQ ID NOs: 18-25. In some embodiments, the
antigen is a plurality of antigens comprising 2, 3, 4, 5, 6, 7 or 8
of the amino acid sequences of any one of SEQ ID NOs 18-25. In some
embodiments, the antigen is a plurality of antigens comprising an
amino acid sequence with at least 90% similarity to SEQ ID NO: 19
and an amino acid sequence with at least 90% similarity to SEQ ID
NO: 23. In a preferred embodiment, the antigen is a plurality of
antigens comprising the amino acid sequence of SEQ ID NO: 19 and
the amino acid sequence of SEQ ID NO: 23. In some embodiments, the
plurality of antigens is contained within a pool of non-covalently
linked peptides. In some embodiments, the plurality of antigens is
contained within a pool of non-covalently linked peptides, wherein
each peptide comprises no more than one antigen. In some
embodiments, the plurality of antigens is contained within a pool
of non-covalently linked peptides, wherein the amino acid sequence
of SEQ ID NO: 19 and the amino acid sequence of SEQ ID NO: 23 are
contained within separate peptides.
[0259] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
modified PBMCs comprise a plurality of antigens that comprise a
plurality of immunogenic epitopes. In further embodiments,
following administration to an individual of the modified PBMCs
comprising the plurality of antigens that comprise the plurality of
immunogenic epitopes, none of the plurality of immunogenic epitopes
decreases an immune response in the individual to any of the other
immunogenic epitopes. In some embodiments, the antigen is a
polypeptide and the immunogenic epitope is an immunogenic peptide
epitope. In some embodiments, the immunogenic peptide epitope is
fused to an N-terminal flanking polypeptide and/or a C-terminal
flanking polypeptide. In some embodiments, the antigen is a
polypeptide comprising an immunogenic peptide epitope and one or
more heterologous peptide sequences. In some embodiments, the
antigen is a polypeptide comprising an immunogenic peptide epitope
that is flanked on the N-terminus and/or the C-terminus by
heterologous peptide sequences. In some embodiments, the flanking
heterologous peptide sequences are derived from disease-associated
immunogenic peptides. In some embodiments, the flanking
heterologous peptide sequences are non-naturally occurring
sequence. In some embodiments, the flanking heterologous peptide
sequences are derived from an immunogenic synthetic long peptide
(SLP). In some embodiments, the N-terminal flanking polypeptide
comprises the amino acid sequence of any one of SEQ ID NOs: 5-10
and/or the C-terminal flanking polypeptide comprises the amino acid
sequence of any one of SEQ ID NOs: 11-17. In some embodiments, the
antigen is capable of being processed into an MHC class
I-restricted peptide and/or an MHC class II-restricted peptide.
Adjuvants
[0260] As used herein, the term "adjuvant" can refer to a substance
which either directly or indirectly modulates and/or engenders an
immune response. In some embodiments of the invention, an adjuvant
is used to condition a population of PBMCs (i.e, the PBMCs are
incubated with an adjuvant prior to administration to an
individual). In some instances, the adjuvant is administered in
conjunction with an antigen to effect enhancement of an immune
response to the antigen as compared to antigen alone. Therefore,
adjuvants can be used to boost elicitation of an immune cell
response (e.g. T cell response) to an antigen. In some embodiments,
the invention provides PBMCs modified to comprise intracellularly
an antigen (such as an HPV antigen) and intracellularly an
adjuvant. In some embodiments, the PBMCs perturbed as described
herein are incubated with both the antigen and an adjuvant.
Exemplary adjuvants include, without limitation, stimulator of
interferon genes (STING) agonists, retinoic acid-inducible gene I
(RIG-I) agonists, and agonists for TLR3, TLR4, TLR7, TLR8 and/or
TLR9. Exemplary adjuvants include, without limitation, CpG ODN,
interferon-.alpha. (IFN-.alpha.), polyinosinic:polycytidylic acid
(polyI:C), imiquimod (R837), resiquimod (R848), or
lipopolysaccharide (LPS). In some embodiments, the adjuvant is CpG
ODN, LPS, IFN-.alpha., STING agonists, RIG-I agonists, poly I:C,
R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR 9 agonist. In
particular embodiments, the adjuvant is a CpG ODN. In some
embodiments, the adjuvant is a CpG ODN. In some embodiments, the
CpG ODN is a Class A CpG ODN, a Class B CpG ODN, or a Class C CpG
ODN. In some embodiments, the CpG ODN adjuvant comprise of a
selection from the group of CpG ODN 1018, CpG ODN 1585, CpG ODN
2216, CpG ODN 2336, CpG ODN 1668, CpG ODN 1826, CPG ODN 2006, CpG
ODN 2007, CpG ODN BW006, CpG ODN D-SL01, CpG ODN 2395, CpG ODN
M362, CpG ODN D-SL03. In some embodiments, the CpG ODN adjuvant is
CpG ODN 1826 (TCCATGACGTTCCTGACGTT (SEQ ID NO: 30)) or CpG ODN 2006
(also known as CpG 7909) (TCGTCGTTTTGTCGTTTTGTCGTT (SEQ ID NO: 31))
oligonucleotide. In some embodiments, the adjuvant is CpG 7909. In
some embodiments, the RIG-I agonist comprises
polyinosinic:polycytidylic acid (polyI:C). Multiple adjuvants can
also be used in conjunction with antigens to enhance the
elicitation of immune response. In some embodiments, the modified
PBMCs comprise more than one adjuvant. Multiple adjuvants can also
be used in conjunction with antigens to enhance the elicitation of
immune response. In some embodiments, the modified PBMCs comprise
more than one adjuvant. In some embodiments, the modified PBMCs
comprise any combination of the adjuvants CpG ODN, LPS,
IFN-.alpha., STING agonists, RIG-I agonists, poly I:C, R837, R848,
a TLR3 agonist, a TLR4 agonist or a TLR 9 agonist.
[0261] In any of the embodiments described herein, unless otherwise
indicated, the adjuvant may refer to (a) an adjuvant that is
incubated with and passes through a perturbed input PBMCs, (b) an
adjuvant incubated with PBMCs for the PBMCs to condition, (c) an
adjuvant co-administered with modified PBMCs to an individual.
[0262] In some embodiments, the concentration of adjuvant incubated
with the perturbed input PBMCs is between about 0.01 .mu.M and
about 10 mM. For example, in some embodiments, the concentration of
adjuvant incubated with the perturbed input PBMCs is any of less
than about 0.01 .mu.M, about 0.1 .mu.M, about 1 .mu.M, about 10
.mu.M, about 100 .mu.M, about 1 mM or about 10 mM. In some
embodiments, the concentration of adjuvant incubated with the
perturbed input PBMCs is greater than about 10 mM. In some
embodiments, the concentration of adjuvant incubated with the
perturbed input PBMCs is any of between about 0.01 .mu.M and about
0.1 .mu.M, between about 0.1 .mu.M and about 1 .mu.M, between about
1 .mu.M and about 10 .mu.M, between about 10 .mu.M and about 100
.mu.M, between about 100 .mu.M and about 1 mM, or between 1 mM and
about 10 mM. In some embodiments, the concentration of adjuvant
incubated with the perturbed input PBMCs is between about 0.1 .mu.M
and about 1 mM. In some embodiments, the concentration of adjuvant
incubated with the perturbed input PBMCs is between about 0.1 .mu.M
and about 10 .mu.M. In some embodiments, the concentration of
adjuvant incubated with the perturbed input PBMCs is 1 .mu.M.
[0263] In some embodiments, the concentration of antigen incubated
with the perturbed input PBMCs is between about 0.01 .mu.M and
about 10 mM. For example, in some embodiments, the concentration of
antigen incubated with the perturbed input PBMCs is any of less
than about 0.01 .mu.M, about 0.1 .mu.M, about 1 .mu.M, about 10
.mu.M, about 100 .mu.M, about 1 mM or about 10 mM. In some
embodiments, the concentration of antigen incubated with the
perturbed input PBMCs is greater than about 10 mM. In some
embodiments, the concentration of antigen incubated with the
perturbed input PBMCs is any of between about 0.01 .mu.M and about
0.1 .mu.M, between about 0.1 .mu.M and about 1 .mu.M, between about
1 .mu.M and about 10 .mu.M, between about 10 .mu.M and about 100
.mu.M, between about 100 .mu.M and about 1 mM, or between 1 mM and
about 10 mM. In some embodiments, the concentration of antigen
incubated with the perturbed input PBMCs is between about 0.1 .mu.M
and about 1 mM. In some embodiments, the concentration of antigen
incubated with the perturbed input PBMCs is between about 0.1 .mu.M
and about 10 .mu.M. In some embodiments, the concentration of
antigen incubated with the perturbed input PBMCs is 1 .mu.M.
[0264] In some embodiments, the molar ratio of antigen to adjuvant
incubated with the perturbed input PBMCs is any of between about
10000:1 to about 1:10000. For example, in some embodiments, the
molar ratio of antigen to adjuvant incubated with the perturbed
input PBMCs is about any of 10000:1, about 1000:1, about 100:1,
about 10:1, about 1:1, about 1:10, about 1:100, about 1:1000, or
about 1:10000. In some embodiments, the molar ratio of antigen to
adjuvant incubated with the perturbed input PBMCs is any of between
about 10000:1 and about 1000:1, between about 1000:1 and about
100:1, between about 100:1 and about 10:1, between about 10:1 and
about 1:1, between about 1:1 and about 1:10, between about 1:10 and
about 1:100, between about 1:100 and about 1:1000, between about
1:1000 and about 1:10000. In some embodiments, the molar ratio of
antigen to adjuvant incubated with the perturbed input PBMCs is
about 200:1. In some embodiments, the molar ratio of antigen to
adjuvant incubated with the perturbed input PBMCs is about
20:1.
[0265] In some embodiments, the modified PBMCs comprise the
adjuvant at a concentration between about 1 nM and about 1 mM. For
example, in some embodiments, the modified PBMCs comprise the
adjuvant at a concentration of any of less than about 0.01 .mu.M,
about 0.1 .mu.M, about 1 .mu.M, about 10 .mu.M, about 100 .mu.M,
about 1 mM or about 10 mM. In some embodiments, the modified PBMCs
comprise the adjuvant at a concentration of greater than about any
of 10 mM. in some embodiments, the modified PBMCs comprise the
adjuvant at a concentration of any of between about 1 nM to about
10 nM, about 0.1 .mu.M and about 1 .mu.M, between about 1 .mu.M and
about 10 .mu.M, between about 10 .mu.M and about 100 .mu.M, between
about 100 .mu.M and about 1 mM, or between 1 mM and about 10 mM. In
some embodiments, the modified PBMCs comprise the adjuvant at a
concentration between about 0.1 .mu.M and about 1 mM. In some
embodiments, the modified PBMCs comprise the adjuvant at a
concentration of about 1 .mu.M.
[0266] In some embodiments, the modified PBMCs comprise the antigen
at a concentration between about 1 nM and about 1 mM. For example,
in some embodiments, the modified PBMCs comprises the antigen at a
concentration of any of less than about 0.01 .mu.M, about 0.1
.mu.M, about 1 .mu.M, about 10 .mu.M, about 100 .mu.M, about 1 mM
or about 10 mM. In some embodiments, the modified PBMCs comprise
the antigen at a concentration of greater than about any of 10 mM.
in some embodiments, the modified PBMCs comprise the antigen at a
concentration of any of between about 1 nM to about 10 nM, about
0.1 .mu.M and about 1 .mu.M, between about 1 .mu.M and about 10
.mu.M, between about 10 .mu.M and about 100 .mu.M, between about
100 .mu.M and about 1 mM, or between 1 mM and about 10 mM. In some
embodiments, the modified PBMCs comprise the antigen at a
concentration between about 0.1 .mu.M and about 1 mM. In some
embodiments, the modified PBMCs comprise the antigen at a
concentration of about 1 .mu.M.
[0267] In some embodiments, the modified PBMCs comprise the nucleic
acid encoding the antigen at a concentration between about 1 nM and
about 1 mM. In some embodiments, the modified PBMCs comprises the
nucleic acid encoding the antigen at a concentration of any of less
than about 0.1 nM, about 1 nM, about 0.01 .mu.M, about 0.1 .mu.M,
about 1 .mu.M, about 10 .mu.M, about 100 .mu.M, about 1 mM or about
10 mM. In some embodiments, the modified PBMCs comprise the nucleic
acid encoding the antigen at a concentration of greater than about
10 mM. In some embodiments, the modified PBMCs comprise the nucleic
acid encoding the antigen at a concentration of any of between
about 0.1 nM to about 1 nM, about 1 nM to about 10 nM, about 10 nM
to about 100 nM, about 0.1 .mu.M and about 1 .mu.M, between about 1
.mu.M and about 10 .mu.M, between about 10 .mu.M and about 100
.mu.M, between about 100 .mu.M and about 1 mM, or between 1 mM and
about 10 mM. In some embodiments, the modified PBMCs comprise the
nucleic acid encoding the antigen at a concentration between about
10 nM and about 100 nM. In some embodiments, the modified PBMCs
comprise the nucleic acid encoding the antigen at a concentration
between about 1 nM and about 10 nM. In some embodiments, the
modified PBMCs comprise the antigen at a concentration of about 50
nM. In some embodiments, the nucleic acid is an mRNA.
[0268] In some embodiments, the modified PBMCs comprise the nucleic
acid encoding the antigen at a concentration between about 0.01
.mu.g/mL to about 10 mg/mL. In some embodiments, the modified PBMCs
comprises the nucleic acid encoding the antigen at a concentration
of any of less than about 0.01 .mu.g/mL, about 0.1 .mu.g/mL, about
1 .mu.g/mL, about 10 .mu.g/mL, about 100 .mu.g/mL, about 1 mg/mL or
about 10 mg/mL. In some embodiments, the modified PBMCs comprise
the nucleic acid encoding the antigen at a concentration of greater
than about 10 .mu.g/mL. in some embodiments, the modified PBMCs
comprise the nucleic acid encoding the antigen at a concentration
of any of between about 0.001 .mu.g/mL to about 0.1 .mu.g/mL, about
0.1 .mu.g/mL and about 1 .mu.g/mL, between about 1 .mu.g/mL and
about 10 .mu.g/mL, between about 10 .mu.g/mL and about 100
.mu.g/mL, between about 100 .mu.g/mL and about 1 mg/mL, or between
1 mg/mL and about 10 mg/mL. In some embodiments, the modified PBMCs
comprise the nucleic acid encoding the antigen at a concentration
between about 0.1 .mu.g/mL and about 1 mg/mL. In some embodiments,
the modified PBMCs comprise the antigen at a concentration of any
one of about 1 .mu.g/mL, about 2 .mu.g/mL, about 5 .mu.g/mL, about
10 .mu.g/mL, about 20 .mu.g/mL, about 25 mg/mL, about 40 .mu.g/mL,
about 50 .mu.g/mL, about 70 .mu.g/mL, about 100 .mu.g/mL, about 200
.mu.g/mL, or about 300 .mu.g/mL, or about 500 .mu.g/mL. In some
embodiments, the nucleic acid is an mRNA.
[0269] In some embodiments, the molar ratio of antigen to adjuvant
in the modified PBMCs is any of between about 10000:1 to about
1:10000. For example, in some embodiments, the molar ratio of
antigen to adjuvant in the modified PBMCs is about any of 10000:1,
about 1000:1, about 100:1, about 10:1, about 1:1, about 1:10, about
1:100, about 1:1000, or about 1:10000. In some embodiments, the
molar ratio of antigen to adjuvant in the modified PBMCs is any of
between about 10000:1 and about 1000:1, between about 1000:1 and
about 100:1, between about 100:1 and about 10:1, between about 10:1
and about 1:1, between about 1:1 and about 1:10, between about 1:10
and about 1:100, between about 1:100 and about 1:1000, between
about 1:1000 and about 1:10000. In some embodiments, the molar
ratio of antigen to adjuvant in the modified PBMCs is about 200:1.
In some embodiments, the molar ratio of antigen to adjuvant in the
modified PBMCs is about 20:1.
[0270] In some embodiments, the antigen complexes with itself, with
other antigens, or with the adjuvant. In some embodiments, the
modified PBMCs comprise a complex comprising: a) the antigen, b)
the antigen and at least one other antigen, and/or c) the antigen
and the adjuvant.
Further Modifications of PBMC Characteristics
[0271] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of modified PBMCs further comprises an agent that
enhances the viability and/or function of the modified PBMCs as
compared to a corresponding plurality of modified PBMCs that does
not comprise the agent. In some embodiments, the plurality of
modified PBMCs further comprises an agent that enhances the
viability and/or function of the modified PBMCs upon freeze-thaw
cycle as compared to a corresponding plurality of modified PBMCs
that does not comprise the agent. In some embodiments, the agent is
a cyropreservation agent and/or a hypothermic preservation agent.
In some embodiments, the cyropreservation agent nor the hypothermic
preservation agent cause not more than 10% or 20% of cell death in
a plurality of PBMCs comprising the agent compared to a
corresponding plurality of PBMCs that does not comprise the agent
before any freeze-thaw cycles. In some embodiments, at least about
70%, about 80%, or about 90% of the plurality of modified PBMCs are
viable after up to 1, 2, 3, 4, 5 freeze-thaw cycles. In some
embodiments, the agent is a compound that enhances endocytosis, a
stabilizing agent or a co-factor. In some embodiments, the agent is
albumin. In some embodiments, the albumin is mouse, bovine, or
human albumin. In some embodiments, the agent is human albumin. In
some embodiments, the agent is one or more of: a divalent metal
cation, glucose, ATP, potassium, glycerol, trehalose, D-sucrose,
PEG1500, L-arginine, L-glutamine, or EDTA. In some embodiments, the
divalent metal cation is one more of Mg.sup.2+, Zn.sup.2+ or
Ca.sup.2+. In some embodiments, the agent is one or more of: sodium
pyruvate, adenine, trehalose, dextrose, mannose, sucrose, human
serum albumin (HSA), DMSO, HEPES, glycerol, glutathione, inosine,
dibasic sodium phosphate, monobasic sodium phosphate, sodium metal
ions, potassium metal ions, magnesium metal ions, chloride,
acetate, gluoconate, sucrose, potassium hydroxide, or sodium
hydroxide. In some embodiments, the agent is one or more of: Sodium
pyruvate, adenine, Rejuvesol.RTM., trehalose, dextrose, mannose,
sucrose, human serum albumin (HSA), PlasmaLyte.RTM., DMSO,
Cryostor.RTM. CS2, Cryostor.RTM. CS5, Cryostor.RTM. CS10,
Cryostor.RTM. CS15, HEPES, glycerol, glutathione,
HypoThermosol.RTM..
[0272] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
modified PBMCs are further modified to increase expression of one
or more of co-stimulatory molecules. In some embodiments, the
co-stimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86),
CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4,
SLAM, CD48, CD58, CD155, or CD112. In some embodiments, the
plurality of modified PBMCs comprises a nucleic acid that results
in increased expression of the one or more co-stimulatory
molecules. In some embodiments, the plurality of modified PBMCs
comprises an mRNA that results in increased expression of the one
or more co-stimulatory molecules. In some embodiments, the
co-stimulatory molecule is a Signal 2 effector in stimulating T
cell activation.
[0273] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
modified PBMCs are further modified to increase expression of one
or more cytokines. In some embodiments, the cytokine is one or more
of IL-2, IL-12, IL-21, or IFN.alpha.2. In some embodiments, the
plurality of modified PBMCs comprises a nucleic acid that results
in increased expression and/or secretion of the one or more
cytokines. In some embodiments, the cytokine is a Signal 3 effector
in stimulating T cell activation.
[0274] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, at
least one cell in the plurality of modified PBMCs is positive for
expression of HLA-A2. In some embodiments, the modified PBMCs
comprise a further modification to modulate MHC class I expression.
In some embodiments, the modified PBMCs comprise a further
modification to modulate expression of HLA-A02 MHC class I. In some
embodiments, the modified PBMCs comprise a further modification to
modulate MHC class II expression. In some embodiments, an innate
immune response mounted in an individual in response to
administration, in an allogeneic context, of the modified PBMCs is
reduced compared to an innate immune response mounted in an
individual in response to administration, in an allogeneic context,
of corresponding modified PBMCs that do not comprise the further
modification. In some embodiments, the circulating half-life of the
modified PBMCs in an individual to which they were administered is
increased compared to the circulating half-life of corresponding
modified PBMCs that do not comprise the further modification in an
individual to which they were administered. In some embodiments,
the circulating half-life of the modified PBMCs in an individual to
which they were administered is increased by about any one of 10%,
25%, 50%, 75%, 100%, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold,
25-fold, 50-fold, 100-fold, 200-fold, or 500-fold or more compared
to the circulating half-life of corresponding modified PBMCs that
do not comprise the further modification in an individual to which
they were administered. In some embodiments, the circulating
half-life of the modified PBMCs in an individual to which they were
administered is essentially the same as the circulating half-life
of corresponding modified PBMCs that do not comprise the further
modification in an individual to which they were administered.
[0275] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
process further comprises a step of incubating the input PBMCs
and/or the modified PBMCs with an agent that enhances the viability
and/or function of the modified PBMCs as compared to corresponding
modified PBMCs prepared without the further incubation step.
Conditioning of PBMCs
[0276] In some embodiments according to any one of methods,
compositions or pluralities of modified PBMCs described herein; the
plurality of modified PBMCs is conditioned. In further embodiments,
the plurality of modified PBMCs is matured. In some embodiments,
the plurality of PBMCs is conditioned subsequent to constriction
mediated delivery. In some embodiments, the plurality of modified
PBMCs comprising the antigen and/or adjuvant is incubated with a
second adjuvant for a sufficient time for the modified PBMCs
comprising the constriction-delivered antigen and/or adjuvant to
condition, thereby generating a conditioned plurality of modified
PBMCs comprising the antigen and/or the adjuvant. In some
embodiments, the plurality of modified PBMCs comprising the antigen
and/or the adjuvant is isolated from the cell suspension before
incubation with the second adjuvant to condition the modified
PBMCs. In some embodiments, the plurality of PBMCs is conditioned
subsequent to constriction mediated delivery. In some embodiments,
the plurality of modified PBMCs comprising the
constriction-delivered antigen and/or adjuvant is incubated with a
second adjuvant for a sufficient time for the modified PBMCs
comprising the constriction-delivered antigen and/or adjuvant to
condition, thereby generating a conditioned plurality of modified
PBMCs comprising the antigen and/or the adjuvant. In some aspects,
there is provided a conditioned plurality of modified PBMCs
comprising an antigen and/or an adjuvant, prepared by a process
comprising the steps of: a) passing a cell suspension comprising a
plurality of input PBMCs through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen and/or the adjuvant
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
and/or the adjuvant for a sufficient time to allow the antigen to
enter the perturbed input PBMCs, thereby generating a plurality of
modified PBMCs comprising the antigen and/or the adjuvant; and c)
incubating the plurality of modified PBMCs comprising the
constriction-delivered antigen and/or adjuvant with a second
adjuvant for a sufficient time for the modified PBMCs comprising
the constriction-delivered antigen and/or adjuvant to condition,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen and/or the adjuvant. In some embodiments,
the process further comprises isolating the plurality of modified
PBMCs comprising the antigen and/or the adjuvant from the cell
suspension before incubation with the second adjuvant to condition
the modified PBMCs. In some embodiments, the constriction-delivered
adjuvant is the same as the conditioning adjuvant. In some
embodiments, the constriction-delivered adjuvant is different from
the conditioning adjuvant.
[0277] In some embodiments, the concentration of antigen incubated
with the modified PBMCs is between about 0.01 .mu.M and about 10
mM. For example, in some embodiments, the concentration of antigen
incubated with the modified PBMCs is any of less than about 0.01
.mu.M, about 0.1 .mu.M, about 1 .mu.M, about 10 .mu.M, about 100
.mu.M, about 1 mM or about 10 mM. In some embodiments, the
concentration of antigen incubated with the modified PBMCs is
greater than about 10 mM. In some embodiments, the concentration of
antigen incubated with the modified PBMCs is any of between about
0.01 .mu.M and about 0.1 .mu.M, between about 0.1 .mu.M and about 1
.mu.M, between about 1 .mu.M and about 10 .mu.M, between about 10
.mu.M and about 100 .mu.M, between about 100 .mu.M and about 1 mM,
or between 1 mM and about 10 mM. In some embodiments, the
concentration of antigen incubated with the modified PBMCs is
between about 0.1 .mu.M and about 1 mM. In some embodiments, the
concentration of antigen incubated with the modified PBMCs is
between about 0.1 .mu.M and about 10 .mu.M. In some embodiments,
the concentration of antigen incubated with the modified PBMCs is 1
.mu.M.
[0278] In some embodiments according to any one of methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of modified PBMCs is incubated with the adjuvant for
about 1 to about 24 hours for the modified PBMCs to condition. In
some embodiments, the plurality of modified PBMCs is incubated with
the adjuvant for about 2 to about 10 hours for the modified PBMCs
to condition. In some embodiments, the plurality of modified PBMCs
is incubated with the adjuvant for about 3 to about 6 hours for the
modified PBMCs to condition. In some embodiments, the plurality of
modified PBMCs is incubated with the adjuvant for any one of about
1 hour, 2 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours,
5.5 hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24
hours for the modified PBMCs to condition. In some embodiments, the
plurality of modified PBMCs is incubated with the adjuvant for
about 4 hours for the modified PBMCs to condition.
[0279] In some embodiments, the plurality of PBMCs is conditioned
prior to constriction mediated delivery. In some embodiments, the
plurality of input PBMCs is incubated with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs. In some
embodiments, there is provided a conditioned plurality of modified
PBMCs comprising an antigen, prepared by a process comprising the
steps of: a) incubating a plurality of input PBMCs with an adjuvant
for a sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; and c) incubating the
conditioned plurality of perturbed input PBMCs with the antigen for
a sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen. In some embodiments, the process
further comprises isolating the conditioned plurality of input
PBMCs from the conditioning adjuvant before passing the conditioned
plurality of input PBMCs through a cell-deforming constriction. In
some embodiments, there is provided a conditioned plurality of
modified PBMCs comprising an antigen and/or an adjuvant, prepared
by a process comprising the steps of: a) incubating a plurality of
input PBMCs with a conditioning adjuvant for a sufficient time for
the input PBMCs to condition, thereby generating a conditioned
plurality of input PBMCs; b) passing a cell suspension comprising
the conditioned plurality of input PBMCs through a cell-deforming
constriction, wherein a diameter of the constriction is a function
of a diameter of the input PBMCs in the suspension, thereby causing
perturbations of the input PBMCs large enough for the antigen
and/or the adjuvant to pass through to form a conditioned plurality
of perturbed input PBMCs; and c) incubating the conditioned
plurality of perturbed input PBMCs with the antigen and/or the
adjuvant for a sufficient time to allow the antigen and/or the
adjuvant to enter the perturbed input PBMCs, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen
and/or the adjuvant. In some embodiments, the process further
comprises isolating the conditioned plurality of input PBMCs from
the conditioning adjuvant before passing the conditioned plurality
of input PBMCs through a cell-deforming constriction. In some
embodiments, the constriction-delivered adjuvant is the same as the
conditioning adjuvant. In some embodiments, the
constriction-delivered adjuvant is different from the conditioning
adjuvant.
[0280] In some embodiments, the concentration of antigen incubated
with the input PBMCs is between about 0.01 .mu.M and about 10 mM.
For example, in some embodiments, the concentration of antigen
incubated with the input PBMCs is any of less than about 0.01
.mu.M, about 0.1 .mu.M, about 1 .mu.M, about 10 .mu.M, about 100
.mu.M, about 1 mM or about 10 mM. In some embodiments, the
concentration of antigen incubated with the input PBMCs is greater
than about 10 mM. In some embodiments, the concentration of antigen
incubated with the input PBMCs is any of between about 0.01 .mu.M
and about 0.1 .mu.M, between about 0.1 .mu.M and about 1 .mu.M,
between about 1 .mu.M and about 10 .mu.M, between about 10 .mu.M
and about 100 .mu.M, between about 100 .mu.M and about 1 mM, or
between 1 mM and about 10 mM. In some embodiments, the
concentration of antigen incubated with the input PBMCs is between
about 0.1 .mu.M and about 1 mM. In some embodiments, the
concentration of antigen incubated with the input PBMCs is between
about 0.1 .mu.M and about 10 .mu.M. In some embodiments, the
concentration of antigen incubated with the input PBMCs is 1
.mu.M.
[0281] In some embodiments according to any one of methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of input PBMCs is incubated with the adjuvant for about 1
to about 24 hours for the input PBMCs to condition. In some
embodiments, the plurality of input PBMCs is incubated with the
adjuvant for about 2 to about 10 hours for the input PBMCs to
condition. In some embodiments, the plurality of input PBMCs is
incubated with the adjuvant for about 3 to about 6 hours for the
input PBMCs to condition. In some embodiments, the plurality of
input PBMCs is incubated with the adjuvant for any one of about 1
hour, 2 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5
hours, 6 hours, 8 hours, 12 hours, 16 hours, 20 hours, or 24 hours
for the input PBMCs to condition. In some embodiments, the
plurality of input PBMCs is incubated with the adjuvant for about 4
hours for the input PBMCs to condition.
[0282] In some embodiments, there is provided a conditioned
plurality of PBMCs comprising an antigen, prepared by incubating
the plurality of PBMCs comprising the antigen with an adjuvant for
a sufficient time for the PBMCs to condition, thereby generating
the conditioned plurality of PBMCs comprising the antigen. In some
embodiments, there is provided a conditioned plurality of PBMCs
comprising an antigen, prepared by incubating the plurality of
PBMCs with an adjuvant for a sufficient time for the PBMCs to
condition prior to introducing the antigen to the PBMCs, thereby
generating the conditioned plurality of PBMCs comprising the
antigen.
[0283] In some embodiments according to any one of the methods,
compositions or pluralities of PBMCs described above, the
concentration of antigen incubated with the PBMCs is between about
0.01 .mu.M and about 10 mM. For example, in some embodiments, the
concentration of antigen incubated with the PBMCs is any of less
than about 0.01 .mu.M, about 0.1 .mu.M, about 1 .mu.M, about 10
.mu.M, about 100 .mu.M, about 1 mM or about 10 mM. In some
embodiments, the concentration of antigen incubated with the PBMCs
is greater than about 10 mM. In some embodiments, the concentration
of antigen incubated with the PBMCs is any of between about 0.01
.mu.M and about 0.1 .mu.M, between about 0.1 .mu.M and about 1
.mu.M, between about 1 .mu.M and about 10 .mu.M, between about 10
.mu.M and about 100 .mu.M, between about 100 .mu.M and about 1 mM,
or between 1 mM and about 10 mM. In some embodiments, the
concentration of antigen incubated with the PBMCs is between about
0.1 .mu.M and about 1 mM. In some embodiments, the concentration of
antigen incubated with the PBMCs is between about 0.1 .mu.M and
about 10 .mu.M. In some embodiments, the concentration of antigen
incubated with the PBMCs is 1 .mu.M.
[0284] In some embodiments according to any of the conditioned
plurality of PBMCs described herein, the plurality of PBMCs is
incubated with the adjuvant for about 1 to about 24 hours for the
PBMCs to condition. In some embodiments, the plurality of PBMCs is
incubated with the adjuvant for about 2 to about 10 hours for the
PBMCs to condition. In some embodiments, the plurality of PBMCs is
incubated with the adjuvant for about 3 to about 6 hours for the
PBMCs to condition. In some embodiments, the plurality of PBMCs is
incubated with the adjuvant for any one of about 1 hour, 2 hours, 3
hours, 3.5 hours, 4 hours, 4.5 hours, 5 hours, 5.5 hours, 6 hours,
8 hours, 12 hours, 16 hours, 20 hours, or 24 hours for the PBMCs to
condition. In some embodiments, the plurality of PBMCs is incubated
with the adjuvant for about 4 hours for the PBMCs to condition.
[0285] In some embodiments according to any one of the conditioned
plurality of PBMCs described herein, one or more co-stimulatory
molecules are upregulated in the conditioned plurality of modified
PBMCs compared to an unconditioned plurality of modified PBMCs. In
some embodiments, one or more co-stimulatory molecules are
upregulated in a subpopulation of cells in the conditioned
plurality of modified PBMCs compared to the subpopulation of cells
in an unconditioned plurality of modified PBMCs. In some
embodiments, one or more co-stimulatory molecules are upregulated
in the B cells of the conditioned plurality of modified PBMCs
compared to the B cells in an unconditioned plurality of modified
PBMCs. In some embodiments, the co-stimulatory molecule is CD80
and/or CD86. In some embodiments, the co-stimulatory molecule is
CD86. In some embodiments, the CD80 and/or CD86 is upregulated in
the B cells of the conditioned plurality of modified PBMCs by more
than about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold,
5-fold, 8-fold, or more than 10-fold compared to the B cells in an
unconditioned plurality of modified PBMCs. In some embodiments, the
CD80 and/or CD86 is upregulated in the B cells of the conditioned
plurality of modified PBMCs by any of about 1.2-fold to about
1.5-fold, about 1.5-fold to about 1.8-fold, about 1.8-fold to about
2-fold, about 2-fold to about 3-fold, about 3-fold to about 4-fold,
about 4-fold to about 5-fold, about 5-fold to about 8-fold, about
8-fold to about 10-fold, about 10-fold to about 20-fold, about
20-fold to about 50-fold, about 50-fold to about 100-fold, about
100-fold to about 200-fold, about 200-fold to about 500-fold, or
more than about 500-fold compared to the B cells in an
unconditioned plurality of modified PBMCs. In some embodiments, the
expression of one or more of IFN-.gamma., IL-6, MCP-1, MIP-1.beta.,
IP-10, or TNF-.alpha. is increased in the conditioned plurality of
modified PBMCs compared to an unconditioned plurality of modified
PBMCs. In some embodiments, the expression of one or more of
IFN-.gamma., IL-6, MCP-1, MIP-1.beta., IP-10, or TNF-.alpha. is
increased a subpopulation of cells in the conditioned plurality
compared to the subpopulation of cells in an unconditioned
plurality of modified PBMCs. In some embodiments, the expression of
one or more of IFN-.gamma., IL-6, MCP-1, MIP-1.beta., IP-10, or
TNF-.alpha. is increased by about 1.2-fold, 1.5-fold, 1.8-fold,
2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold in the
conditioned plurality of modified PBMCs compared to an
unconditioned plurality of modified PBMCs. In some embodiments, the
expression of one or more of IFN-.gamma., IL-6, MCP-1, MIP-1.beta.,
IP-10, or TNF-.alpha. is increased by any of about 1.2-fold to
about 1.5-fold, about 1.5-fold to about 1.8-fold, about 1.8-fold to
about 2-fold, about 2-fold to about 3-fold, about 3-fold to about
4-fold, about 4-fold to about 5-fold, about 5-fold to about 8-fold,
about 8-fold to about 10-fold, about 10-fold to about 20-fold,
about 20-fold to about 50-fold, about 50-fold to about 100-fold,
about 100-fold to about 200-fold, about 200-fold to about 500-fold,
or more than about 500-fold in the conditioned plurality of
modified PBMCs compared to an unconditioned plurality of modified
PBMCs.
[0286] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs. In some embodiments, the plurality of
PBMCs comprises a nucleic acid encoding an antigen. In some
embodiments, the plurality of PBMCs comprises an mRNA encoding an
antigen.
Microfluidic Systems and Components Thereof
Microfluidic Channels to Provide Cell-Deforming Constrictions
[0287] In some embodiments, the invention provides methods for
modulating an immune response by passing a cell suspension
comprising a PBMCs through a constriction, wherein the constriction
deforms the PBMCs thereby causing a perturbation of the PBMCs such
that an antigen and/or adjuvant enters the PBMCs, wherein the
constriction is contained within a microfluidic channel. In some
embodiments, multiple constrictions can be placed in parallel
and/or in series within the microfluidic channel. Exemplary
microfluidic channels containing cell-deforming constrictions for
use in the methods disclosed herein are described in WO2013059343.
Exemplary surfaces having pores for use in the methods disclosed
herein are described in WO2017041050.
[0288] In some embodiments, the microfluidic channel includes a
lumen and is configured such that PBMCs suspended in a buffer can
pass through, wherein the microfluidic channel includes a
constriction. The microfluidic channel can be made of any one of a
number of materials, including silicon, metal (e.g., stainless
steel), plastic (e.g., polystyrene), ceramics, glass, crystalline
substrates, amorphous substrates, or polymers (e.g., Poly-methyl
methacrylate (PMMA), PDMS, Cyclic Olefin Copolymer (COC), etc.).
Fabrication of the microfluidic channel can be performed by any
method known in the art, including dry etching, wet etching,
photolithography, injection molding, laser ablation, or SU-8
masks.
[0289] In some embodiments, the constriction within the
microfluidic channel includes an entrance portion, a centerpoint,
and an exit portion. In some embodiments, the length, depth, and
width of the constriction within the microfluidic channel can vary.
In some embodiments, the diameter of the constriction within the
microfluidic channel is a function of the diameter of the input
PBMCs. Methods to determine the diameter of a PBMC are known in the
art; for example, high-content imaging, cell counters or flow
cytometry. In some embodiments, the diameter of the constriction
within the microfluidic channel is about 20%, to about 99% of the
mean diameter of the plurality of input PBMCs. In some embodiments,
the constriction size is about 20%, about 30%, about 40%, about
50%, about 60%, about 70%, about 80%, about 90%, or about 99% of
the mean diameter of PBMCs or mean diameter of a subpopulation of
PBMCs. In some embodiments, the constriction size is about 20%,
about 30%, about 40%, about 50%, about 60%, about 70%, about 80%,
about 90%, or about 99% of the mean of minimum cross-sectional
distance of the plurality of input PBMCs. In some embodiments, the
channel comprises a constriction width of between about 2 .mu.m and
about 10 .mu.m or any width or range of widths therebetween. In
some embodiments, the channel comprises a constriction width of
between about 3 .mu.m and about 10 .mu.m. In some embodiments, the
channel comprises a constriction width of between about 3 .mu.m and
about 6 .mu.m. In some embodiments, the channel comprises a
constriction width of between about 4.2 .mu.m and about 4.8 .mu.m.
For example, the constriction width can be any one of about 2
.mu.m, about 2.5 .mu.m, about 3 .mu.m, about 3.5 .mu.m, about 4
.mu.m, about 4.5 .mu.m, about 5 .mu.m, about 5.5 .mu.m, about 6
.mu.m, about 6.5 .mu.m, or about 7 .mu.m. In some embodiments, the
channel comprises a constriction length of about 10 .mu.m and a
constriction width of about 3.5 .mu.m. In some embodiments, the
channel comprises a constriction length of about 10 .mu.m and a
constriction width of about 4 .mu.m. In some embodiments, the
channel comprises a constriction length of about 10 .mu.m and a
constriction width of about 4.5 .mu.m. The cross-section of the
channel, the entrance portion, the centerpoint, and the exit
portion can also vary. For example, the cross-sections can be
circular, elliptical, an elongated slit, square, hexagonal, or
triangular in shape. The entrance portion defines a constriction
angle, wherein the constriction angle is optimized to reduce
clogging of the channel and optimized for enhanced delivery of a
compound into the PBMCs. The angle of the exit portion can vary as
well. For example, the angle of the exit portion is configured to
reduce the likelihood of turbulence that can result in non-laminar
flow. In some embodiments, the walls of the entrance portion and/or
the exit portion are linear. In other embodiments, the walls of the
entrance portion and/or the exit portion are curved. Surface having
pores to provide cell-deforming constrictions
[0290] In some embodiments, the invention provides methods for
modulating an immune response by passing a cell suspension
comprising a plurality of PBMCs through a constriction, wherein the
constriction deforms the PBMCs thereby causing a perturbation of
the PBMCs such that an antigen and/or adjuvant enters the PBMCs,
wherein the constriction is a pore or contained within a pore. In
some embodiments, the pore is contained in a surface. Exemplary
surfaces having pores for use in the methods disclosed herein are
described in WO2017041050.
[0291] The surfaces as disclosed herein can be made of any one of a
number of materials and take any one of a number of forms. In some
embodiments, the surface is a filter. In some embodiments, the
surface is a membrane. In some embodiments, the filter is a
tangential flow filter. In some embodiments, the surface is a
sponge or sponge-like matrix. In some embodiments, the surface is a
matrix.
[0292] In some embodiments the surface is a tortuous path surface.
In some embodiments, the tortuous path surface comprises cellulose
acetate. In some embodiments, the surface comprises a material
selected from, without limitation, synthetic or natural polymers,
polycarbonate, silicon, glass, metal, alloy, cellulose nitrate,
silver, cellulose acetate, nylon, polyester, polyethersulfone,
polyacrylonitrile (PAN), polypropylene, PVDF,
polytetrafluorethylene, mixed cellulose ester, porcelain, and
ceramic.
[0293] The surface disclosed herein can have any shape known in the
art; e.g. a 3-dimensional shape. The 2-dimensional shape of the
surface can be, without limitation, circular, elliptical, round,
square, star-shaped, triangular, polygonal, pentagonal, hexagonal,
heptagonal, or octagonal. In some embodiments, the surface is round
in shape. In some embodiments, the surface 3-dimensional shape is
cylindrical, conical, or cuboidal.
[0294] The surface can have various cross-sectional widths and
thicknesses. In some embodiments, the surface cross-sectional width
is between about 1 mm and about 1 m or any cross-sectional width or
range of cross-sectional widths therebetween. In some embodiments,
the surface has a defined thickness. In some embodiments, the
surface thickness is uniform. In some embodiments, the surface
thickness is variable. For example, in some embodiments, portions
of the surface are thicker or thinner than other portions of the
surface. In some embodiments, the surface thickness varies by about
1% to about 90% or any percentage or range of percentages
therebetween. In some embodiments, the surface is between about
0.01 .mu.m to about 5 mm thick or any thickness or range of
thicknesses therebetween.
[0295] The entrances and exits of the pore passage may have a
variety of angles. The pore angle can be selected to minimize
clogging of the pore while PBMCs are passing through. In some
embodiments the flow rate through the surface is between about
0.001 mL/cm.sup.2/sec to about 100 L/cm.sup.2/sec or any rate or
range of rates therebetween. For example, the angle of the entrance
or exit portion can be between about 0 and about 90 degrees. In
some embodiments, the entrance or exit portion can be greater than
90 degrees. In some embodiments, the pores have identical entrance
and exit angles. In some embodiments, the pores have different
entrance and exit angles. In some embodiments, the pore edge is
smooth, e.g. rounded or curved. A smooth pore edge has a
continuous, flat, and even surface without bumps, ridges, or uneven
parts. In some embodiments, the pore edge is sharp. A sharp pore
edge has a thin edge that is pointed or at an acute angle. In some
embodiments, the pore passage is straight. A straight pore passage
does not contain curves, bends, angles, or other irregularities. In
some embodiments, the pore passage is curved. A curved pore passage
is bent or deviates from a straight line. In some embodiments, the
pore passage has multiple curves, e.g. about 2, 3, 4, 5, 6, 7, 8,
9, 10 or more curves.
[0296] The pores can have any shape known in the art, including a
2-dimensional or 3-dimensional shape. The pore shape (e.g., the
cross-sectional shape) can be, without limitation, circular,
elliptical, round, square, star-shaped, triangular, polygonal,
pentagonal, hexagonal, heptagonal, and octagonal. In some
embodiments, the cross-section of the pore is round in shape. In
some embodiments, the 3-dimensional shape of the pore is
cylindrical or conical. In some embodiments, the pore has a fluted
entrance and exit shape. In some embodiments, the pore shape is
homogenous (i.e. consistent or regular) among pores within a given
surface. In some embodiments, the pore shape is heterogeneous (i.e.
mixed or varied) among pores within a given surface.
[0297] The surfaces described herein can have a range of total pore
numbers. In some embodiments, the pores encompass about 10% to
about 80% of the total surface area. In some embodiments, the
surface contains about 1.0.times.10.sup.5 to about
1.0.times.10.sup.30 total pores or any number or range of numbers
therebetween. In some embodiments, the surface comprises between
about 10 and about 1.0.times.10.sup.15 pores/mm.sup.2 surface
area.
[0298] The pores can be distributed in numerous ways within a given
surface. In some embodiments, the pores are distributed in parallel
within a given surface. In one such example, the pores are
distributed side-by-side in the same direction and are the same
distance apart within a given surface. In some embodiments, the
pore distribution is ordered or homogeneous. In one such example,
the pores are distributed in a regular, systematic pattern or are
the same distance apart within a given surface. In some
embodiments, the pore distribution is random or heterogeneous. In
one such example, the pores are distributed in an irregular,
disordered pattern or are different distances apart within a given
surface. In some embodiments, multiple surfaces are distributed in
series. The multiple surfaces can be homogeneous or heterogeneous
in surface size, shape, and/or roughness. The multiple surfaces can
further contain pores with homogeneous or heterogeneous pore size,
shape, and/or number, thereby enabling the simultaneous delivery of
a range of compounds into different PBMC types.
[0299] In some embodiments, an individual pore has a uniform width
dimension (i.e. constant width along the length of the pore
passage). In some embodiments, an individual pore has a variable
width (i.e. increasing or decreasing width along the length of the
pore passage). In some embodiments, pores within a given surface
have the same individual pore depths. In some embodiments, pores
within a given surface have different individual pore depths. In
some embodiments, the pores are immediately adjacent to each other.
In some embodiments, the pores are separated from each other by a
distance. In some embodiments, the pores are separated from each
other by a distance of about 0.001 .mu.m to about 30 mm or any
distance or range of distances therebetween.
[0300] In some embodiments, the surface is coated with a material.
The material can be selected from any material known in the art,
including, without limitation, Teflon, an adhesive coating,
surfactants, proteins, adhesion molecules, antibodies,
anticoagulants, factors that modulate cellular function, nucleic
acids, lipids, carbohydrates, or transmembrane proteins. In some
embodiments, the surface is coated with polyvinylpyrrolidone (PVP).
In some embodiments, the material is covalently attached to the
surface. In some embodiments, the material is non-covalently
attached or adsorbed to the surface. In some embodiments, the
surface molecules are released as the PBMCs pass through the
pores.
[0301] In some embodiments, the surface has modified chemical
properties. In some embodiments, the surface is polar. In some
embodiments, the surface is hydrophilic. In some embodiments, the
surface is non-polar. In some embodiments, the surface is
hydrophobic. In some embodiments, the surface is charged. In some
embodiments, the surface is positively and/or negatively charged.
In some embodiments, the surface can be positively charged in some
regions and negatively charged in other regions. In some
embodiments, the surface has an overall positive or overall
negative charge. In some embodiments, the surface can be any one of
smooth, electropolished, rough, or plasma treated. In some
embodiments, the surface comprises a zwitterion or dipolar
compound. In some embodiments, the surface is plasma treated.
[0302] In some embodiments, the surface is contained within a
larger module. In some embodiments, the surface is contained within
a syringe, such as a plastic or glass syringe. In some embodiments,
the surface is contained within a plastic filter holder. In some
embodiments, the surface is contained within a pipette tip.
[0303] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs. In some embodiments, the one or more
nucleic acids are carried in one or more vehicles, wherein the one
or more vehicles are delivered to the input PBMCs. In some
embodiments, the vehicle is a virus or a viral-associated particle.
In some embodiments, the virus comprises one or more of: an
adenovirus, an adeno-associated virus (AAV), a baculovirus, a
herpes virus, or a retrovirus. In some embodiments, the virus
comprises an AAV. In some embodiments, the vehicle is a lipid-based
vehicle, e.g., a liposome. In some embodiments, the vehicle is a
nanoparticle.
Cell Perturbations
[0304] In some embodiments, the invention provides methods for
modulating an immune response by passing a cell suspension
comprising PBMCs through a constriction, wherein the constriction
deforms the PBMCs thereby causing a perturbation of the PBMCs such
that an antigen and/or adjuvant enters the PBMCs, wherein the
perturbation in the PBMCs is a breach in the PBMCs that allows
material from outside the PBMCs to move into the PBMCs (e.g., a
hole, tear, cavity, aperture, pore, break, gap, perforation). The
deformation can be caused by, for example, mechanical strain or
mechanical strain and shear forces. In some embodiments, the
perturbation is a perturbation within the PBMCs cell membranes. In
some embodiments, the perturbation is transient. In some
embodiments, the PBMCs perturbation lasts from about
1.0.times.10.sup.-9 seconds to about 2 hours, or any time or range
of times therebetween. In some embodiments, the PBMCs perturbation
lasts for about 1.0.times.10.sup.-9 second to about 1 second, about
1 second to about 1 minute, or about 1 minute to about 1 hour. In
some embodiments, the PBMCs perturbation lasts for between any one
of about 1.0.times.10.sup.-9 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-2, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-3, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-4, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-5, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-6, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-7, or about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-8 seconds. In some
embodiment, the PBMCs perturbation lasts for any one of about
1.0.times.10.sup.-8 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-7 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-6 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-5 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-4 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-3 to about 1.0.times.10.sup.-1, or about
1.0.times.10.sup.-2 to about 1.0.times.10.sup.-1 seconds. The PBMCs
perturbations (e.g., pores or holes) created by the methods
described herein are not formed as a result of assembly of protein
subunits to form a multimeric pore structure such as that created
by complement or bacterial hemolysins.
[0305] As the PBMCs passes through the constriction, the
constriction temporarily imparts injury to the PBMCs membranes that
allows for passive diffusion of material through the perturbation.
In some embodiments, the PBMCs are only deformed for a brief period
of time, on the order of 100 .mu.s to minimize the chance of
activating apoptotic pathways through cell signaling mechanisms,
although other durations are possible (e.g., ranging from
nanoseconds to hours). In some embodiments, the PBMCs are deformed
for about 1.0.times.10.sup.-9 seconds to about 2 hours, or any time
or range of times therebetween. In some embodiments, the PBMCs are
deformed for about 1.0.times.10.sup.-9 second to about 1 second,
about 1 second to about 1 minute, or about 1 minute to about 1
hour. In some embodiments, the PBMCs are deformed for between any
one of about 1.0.times.10.sup.-9 to about 1.0.times.10.sup.-1,
about 1.0.times.10.sup.-9 to about 1.0.times.10.sup.-2, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-3, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-4, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-5, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-6, about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-7, or about
1.0.times.10.sup.-9 to about 1.0.times.10.sup.-5 seconds. In some
embodiment, the PBMCs are deformed for any one of about
1.0.times.10.sup.-8 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-7 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-6 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-5 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-4 to about 1.0.times.10.sup.-1, about
1.0.times.10.sup.-3 to about 1.0.times.10.sup.-1, or about
1.0.times.10.sup.-2 to about 1.0.times.10.sup.-1 seconds. In some
embodiments, deforming the PBMCs includes deforming the PBMCs for a
time ranging from, without limitation, about 1 .mu.s to at least
about 750 .mu.s, e.g., at least about 1 .mu.s, 10 .mu.s, 50 .mu.s,
100 .mu.s, 500 .mu.s, or 750 .mu.s.
[0306] In some embodiments, the passage of the antigen and/or
adjuvant into the PBMCs occurs simultaneously with the PBMCs
passing through the constriction and/or the perturbation of the
PBMCs. In some embodiments, passage of the compound into the PBMCs
occurs after the PBMCs pass through the constriction. In some
embodiments, passage of the compound into the PBMCs occurs on the
order of minutes after the PBMCs pass through the constriction. In
some embodiments, the passage of the compound into the PBMCs occurs
from about 1.0.times.10.sup.-2 seconds to at least about 30 minutes
after the PBMCs pass through the constriction. For example, the
passage of the compound into the PBMCs occurs from about
1.0.times.10.sup.-2 seconds to about 1 second, about 1 second to
about 1 minute, or about 1 minute to about 30 minutes after the
PBMCs pass through the constriction. In some embodiments, the
passage of the compound into the PBMCs occurs about
1.0.times.10.sup.-2 seconds to about 10 minutes, about
1.0.times.10.sup.-2 seconds to about 5 minutes, about
1.0.times.10.sup.-2 seconds to about 1 minute, about
1.0.times.10.sup.-2 seconds to about 30 seconds, about
1.0.times.10.sup.-2 seconds to about 10 seconds, about
1.0.times.10.sup.-2 seconds to about 1 second, or about
1.0.times.10.sup.-2 seconds to about 0.1 second after the PBMCs
passes through the constriction. In some embodiments, the passage
of the compound into the PBMCs occurs about 1.0.times.10.sup.-1
seconds to about 10 minutes, about 1 second to about 10 minutes,
about 10 seconds to about 10 minutes, about 50 seconds to about 10
minutes, about 1 minute to about 10 minutes, or about 5 minutes to
about 10 minutes after the PBMCs pass through the constriction. In
some embodiments, a perturbation in the PBMCs after they pass
through the constriction is corrected within the order of about
five minutes after the PBMCs pass through the constriction.
[0307] In some embodiments, the cell viability after passing
through a constriction is about 5% to about 100%. In some
embodiments, the cell viability after passing through the
constriction is at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%,
70%, 75%, 80%, 85%, 90%, 95%, or 99%. In some embodiments, the cell
viability is measured from about 1.0.times.10.sup.-2 seconds to at
least about 10 days after the PBMCs pass through the constriction.
For example, the cell viability is measured from about
1.0.times.10.sup.-2 seconds to about 1 second, about 1 second to
about 1 minute, about 1 minute to about 30 minutes, or about 30
minutes to about 2 hours after the PBMCs pass through the
constriction. In some embodiments, the cell viability is measured
about 1.0.times.10.sup.-2 seconds to about 2 hours, about
1.0.times.10.sup.-2 seconds to about 1 hour, about
1.0.times.10.sup.-2 seconds to about 30 minutes, about
1.0.times.10.sup.-2 seconds to about 1 minute, about
1.0.times.10.sup.-2 seconds to about 30 seconds, about
1.0.times.10.sup.-2 seconds to about 1 second, or about
1.0.times.10.sup.-2 seconds to about 0.1 second after the PBMCs
pass through the constriction. In some embodiments, the cell
viability is measured about 1.5 hours to about 2 hours, about 1
hour to about 2 hours, about 30 minutes to about 2 hours, about 15
minutes to about 2 hours, about 1 minute to about 2 hours, about 30
seconds to about 2 hours, or about 1 second to about 2 hours after
the PBMCs pass through the constriction. In some embodiments, the
cell viability is measured about 2 hours to about 5 hours, about 5
hours to about 12 hours, about 12 hours to about 24 hours, or about
24 hours to about 10 days after the PBMCs pass through the
constriction.
Delivery Parameters
[0308] A number of parameters may influence the delivery of a
compound to PBMCs for modulating an immune response by the methods
described herein. In some embodiments, the cell suspension is
contacted with the compound before, concurrently, or after passing
through the constriction. The PBMCs may pass through the
constriction suspended in a solution that includes the compound to
deliver, although the compound can be added to the cell suspension
after the PBMCs pass through the constriction. In some embodiments,
the compound to be delivered is coated on the constriction.
[0309] Examples of parameters that may influence the delivery of
the compound into the PBMCs include, but are not limited to, the
dimensions of the constriction, the entrance angle of the
constriction, the surface properties of the constrictions (e.g.,
roughness, chemical modification, hydrophilic, hydrophobic, etc.),
the operating flow speeds (e.g., cell transit time through the
constriction), the PBMC concentration, the concentration of the
compound in the cell suspension, and the amount of time that the
PBMCs recover or incubates after passing through the constrictions
can affect the passage of the delivered compound into the PBMCs.
Additional parameters influencing the delivery of the compound into
the PBMCs can include the velocity of the PBMCs in the
constriction, the shear rate in the constriction, the viscosity of
the cell suspension, the velocity component that is perpendicular
to flow velocity, and time in the constriction. In addition,
multiple chips comprising channels in series and/or in parallel may
impact delivery to PBMC. Multiple chips in parallel may be useful
to enhance throughput. Such parameters can be designed to control
delivery of the compound. In some embodiments, the PBMCs
concentration ranges from about 10 to at least about 10.sup.12
cells/mL or any concentration or range of concentrations
therebetween. In some embodiments, delivery compound concentrations
can range from about 10 .mu.g/mL to about 1 g/mL or any
concentration or range of concentrations therebetween. In some
embodiments, delivery compound concentrations can range from about
1 pM to at least about 2 M or any concentration or range of
concentrations therebetween.
[0310] The temperature used in the methods of the present
disclosure can be adjusted to affect compound delivery and cell
viability. In some embodiments, the method is performed between
about -5.degree. C. and about 45.degree. C. For example, the
methods can be carried out at room temperature (e.g., about
20.degree. C.), physiological temperature (e.g., about 37.degree.
C.), higher than physiological temperature (e.g., greater than
about 37.degree. C. to 45.degree. C. or more), or reduced
temperature (e.g., about -5.degree. C. to about 4.degree. C.), or
temperatures between these exemplary temperatures.
[0311] Various methods can be utilized to drive the PBMCs through
the constrictions. For example, pressure can be applied by a pump
on the entrance side (e.g., compressor), a vacuum can be applied by
a vacuum pump on the exit side, capillary action can be applied
through a tube, and/or the system can be gravity fed. Displacement
based flow systems can also be used (e.g., syringe pump,
peristaltic pump, manual syringe or pipette, pistons, etc.). In
some embodiments, the PBMCs are passed through the constrictions by
positive pressure or negative pressure. In some embodiments, the
PBMCs are passed through the constrictions by constant pressure or
variable pressure. In some embodiments, pressure is applied using a
syringe. In some embodiments, the pressure is positive pressure
applied using a gas (e.g., from a gas cylinder). In some
embodiments, pressure is applied using a pump. In some embodiments,
the pump is a peristaltic pump or a diaphragm pump. In some
embodiments, pressure is applied using a vacuum. In some
embodiments, the PBMCs are passed through the constrictions by
g-force. In some embodiments, the PBMCs are passed through the
constrictions by centrifugal force. In some embodiments, the PBMCs
are passed through the constrictions by capillary pressure.
[0312] In some embodiments, fluid flow directs the PBMCs through
the constrictions. In some embodiments, the fluid flow is turbulent
flow prior to the PBMCs passing through the constriction. Turbulent
flow is a fluid flow in which the velocity at a given point varies
erratically in magnitude and direction. In some embodiments, the
fluid flow through the constriction is laminar flow. Laminar flow
involves uninterrupted flow in a fluid near a solid boundary in
which the direction of flow at every point remains constant. In
some embodiments, the fluid flow is turbulent flow after the PBMCs
pass through the constriction. The velocity at which the PBMCs pass
through the constrictions can be varied. In some embodiments, the
PBMCs pass through the constrictions at a uniform cell speed. In
some embodiments, the PBMCs pass through the constrictions at a
fluctuating cell speed.
[0313] In other embodiments, a combination treatment is used to
modulate an immune response by passing a cell suspension comprising
PBMCs through a constriction, wherein the constriction deforms the
PBMCs thereby causing a perturbation of the PBMCs such that an
antigen and/or adjuvant enters the PBMCs, e.g., the methods
described herein, followed by exposure to an electric field
downstream of the constriction. In some embodiments, the PBMCs are
passed through an electric field generated by at least one
electrode after passing through the constriction. In some
embodiments, the electric field assists in delivery of compounds to
a second location inside the PBMCs such as the PBMCs nuclei. For
example, the combination of a cell-deforming constriction and an
electric field delivers a plasmid encoding an antibody into the
PBMCs (e.g., the cell nucleus), resulting in the de novo production
of antibody. In some embodiments, one or more electrodes are in
proximity to the cell-deforming constriction to generate an
electric field. In some embodiments, the electric field is between
about 0.1 kV/m to about 100 MV/m, or any number or range of numbers
therebetween. In some embodiments, an integrated circuit is used to
provide an electrical signal to drive the electrodes. In some
embodiments, the PBMCs are exposed to the electric field for a
pulse width of between about 1 ns to about 1 s and a period of
between about 100 ns to about 10 s or any time or range of times
therebetween.
Cell Suspensions for Delivery to PBMCs
[0314] The cell suspension may be a mixed or purified population or
plurality of PBMCs. In some embodiments, the cell suspension is a
mixed cell population, such as whole blood. In some embodiments,
the cell suspension is a purified cell population, such as a
purified population (e.g., plurality) of PBMCs. In other
embodiments, the population (e.g., plurality) of PBMCs is depleted
of one or more cells. In some embodiments, the population of PBMCs
is depleted of one or more of T cells, B cells, NK cells,
macrophages or dendritic cells.
[0315] The composition of the cell suspension (e.g., osmolarity,
salt concentration, serum content, cell concentration, pH, etc.)
can impact delivery of the compound for modulating an immune
response. In some embodiments, the suspension comprises whole
blood. Alternatively, the cell suspension is a mixture of cells in
a physiological saline solution or physiological medium other than
blood. In some embodiments, the cell suspension comprises an
aqueous solution. In some embodiments, the aqueous solution
comprises cell culture medium, phosphate buffered saline (PBS),
salts, metal ions, sugars, growth factors, animal derived products,
bulking materials, surfactants, lubricants, lipids, vitamins, amino
acids, proteins, cell cycle inhibitors, and/or an agent that
impacts actin polymerization. In some embodiments, the cell culture
medium is DMEM, Opti-MEM.RTM., IMDM, RPMI, X-Vivo 10.TM., and
X-Vivo 15.TM.. Additionally, solution buffer can include one or
more lubricants (Pluronics.RTM. or other surfactants) that can be
designed, for example, to reduce or eliminate clogging of the
constriction or pore and improve cell viability. Exemplary
surfactants include, without limitation, poloxamer, polysorbates,
sugars or sugar alcohols such as mannitol, sorbitol, animal derived
serum, and albumin protein.
[0316] In some configurations with certain types of PBMCs, the
PBMCs can be incubated in one or more solutions that aid in the
delivery of the compound to the interior of the PBMCs. In some
embodiments, the aqueous solution comprises an agent that impacts
actin polymerization. In some embodiments, the agent that impacts
actin polymerization is Latrunculin A, Cytochalasin, and/or
Colchicine. For example, the PBMCs can be incubated in a
depolymerization solution such as Lantrunculin A (0.1 .mu.g/mL) for
1 hour prior to delivery to depolymerize the actin cytoskeleton. As
an additional example, the PBMCs can be incubated in 10 .mu.M
Colchicine (Sigma) for 2 hours prior to delivery to depolymerize
the microtubule network.
[0317] The viscosity of the cell suspension can also impact the
methods disclosed herein. In some embodiments, the viscosity of the
cell suspension ranges from about 8.9.times.10.sup.-4 Pas to about
4.0.times.10.sup.-3 Pas or any value or range of values
therebetween. In some embodiments, the viscosity ranges between any
one of about 8.9.times.10.sup.-4 Pas to about 4.0.times.10.sup.-3
Pas, about 8.9.times.10.sup.-4 Pas to about 3.0.times.10.sup.-3
Pas, about 8.9.times.10.sup.-4 Pas to about 2.0.times.10.sup.-3
Pas, or about 8.9.times.10.sup.-3 Pas to about 1.0.times.10.sup.-3
Pas. In some embodiments, the viscosity ranges between any one of
about 0.89 cP to about 4.0 cP, about 0.89 cP to about 3.0 cP, about
0.89 cP to about 2.0 cP, or about 0.89 cP to about 1.0 cP. In some
embodiments, a shear thinning effect is observed, in which the
viscosity of the cell suspension decreases under conditions of
shear strain. Viscosity can be measured by any method known in the
art, including without limitation, viscometers, such as a glass
capillary viscometer, or rheometers. A viscometer measures
viscosity under one flow condition, while a rheometer is used to
measure viscosities which vary with flow conditions. In some
embodiments, the viscosity is measured for a shear thinning
solution such as blood. In some embodiments, the viscosity is
measured between about -5.degree. C. and about 45.degree. C. For
example, the viscosity is measured at room temperature (e.g., about
20.degree. C.), physiological temperature (e.g., about 37.degree.
C.), higher than physiological temperature (e.g., greater than
about 37.degree. C. to 45.degree. C. or more), reduced temperature
(e.g., about -5.degree. C. to about 4.degree. C.), or temperatures
between these exemplary temperatures.
Constriction Mediated Delivery
[0318] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
diameter of the constriction is a function of a diameter of the
PBMCs, such as the mean diameter of a plurality of PBMCs, or a mean
diameter of a subpopulation within plurality of the PBMCs. In some
embodiments, the diameter of a cell is measured by the minimum
cross-sectional distance of the cell (e.g. a cell within the
plurality of PBMCs).
[0319] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
diameter of the constriction is about 10% to about 99% of the mean
diameter of the plurality of input PBMCs. In some embodiments, the
diameter of the constriction is any one of about 10% to about 90%,
about 10% to about 80%, about 10% to about 70%, about 20% to about
60%, about 40% to about 60%, or about 30% to about 45% of the mean
diameter of the plurality of input PBMCs. In some embodiments, the
diameter of the constriction is any one of about 10% to about 20%,
about 20% to about 30%, about 30% to about 40%, about 40% to about
50%, about 50% to about 60%, about 60% to about 70%, about 70% to
about 80%, about 80% to about 90%, or about 90% to about 99% of the
mean diameter of the plurality of input PBMCs. In some embodiments,
the diameter of the constriction is any one of about 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 99% of the mean diameter of the plurality of input
PBMCs.
[0320] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
diameter of the constriction is about 10% to about 99% of the mean
diameter of a subpopulation of cells having the smallest diameter
within the plurality of input PBMCs. In some embodiments, the
diameter of the constriction is any one of about 10% to about 90%,
about 10% to about 80%, about 10% to about 70%, about 20% to about
60%, about 40% to about 60%, about 30% to about 45%, about 50% to
about 99%, about 50% to about 90%, about 50% to about 80%, about
50% to about 70%, about 60% to about 90%, about 60% to about 80%,
or about 60% to about 70% of the mean diameter of a subpopulation
of cells having the smallest diameter within the plurality of input
PBMCs. In some embodiments, the diameter of the constriction is any
one of about 10% to about 20%, about 20% to about 30%, about 30% to
about 40%, about 40% to about 50%, about 50% to about 60%, about
60% to about 70%, about 70% to about 80%, about 80% to about 90%,
or about 90% to about 99% of the mean diameter of a subpopulation
of cells having the smallest diameter within the plurality of input
PBMCs. In some embodiments, the diameter of the constriction is any
one of about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the mean diameter of a
subpopulation of cells having the smallest diameter within the
plurality of input PBMCs. In some embodiments, the subpopulation of
cells having the smallest mean diameter within the plurality of
input PBMCs is a population of lymphocytes, wherein the diameter of
the population of lymphocytes is about 6 .mu.m to about 10 .mu.m.
In some embodiments, the mean diameter of the population of
lymphocytes is about 7 .mu.m. In some embodiments, the population
of lymphocytes is a population of T cells. In some embodiments, the
lymphocytes are T cells. In some embodiments, the subpopulation of
cells having the smallest mean diameter within the plurality of
input PBMCs are T cells.
[0321] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
diameter of the constriction is about 10% to about 99% of the mean
diameter of a subpopulation of cells having the largest diameter
within the plurality of input PBMCs. In some embodiments, the
diameter of the constriction is any one of about 10% to about 90%,
about 10% to about 80%, about 10% to about 70%, about 20% to about
60%, about 40% to about 60%, about 30% to about 45%, about 15% to
about 30%, about 15% to about 20%, about 20% to about 25%, about
25% to about 30%, about 20% to about 30%, about 30% to about 70%,
or about 30% to about 60% of the mean diameter of a subpopulation
of cells having the largest diameter within the plurality of input
PBMCs. In some embodiments, the diameter of the constriction is any
one of about 5% to about 10%, about 10% to about 20%, about 20% to
about 30%, about 30% to about 40%, about 40% to about 50%, about
50% to about 60%, about 60% to about 70%, about 70% to about 80%,
about 80% to about 90%, or about 90% to about 99% of the mean
diameter of a subpopulation of cells having the largest diameter
within the plurality of input PBMCs. In some embodiments, the
diameter of the constriction is any one of about 5%, 10%, 15%, 20%,
25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,
90%, 95%, or 99% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input PBMCs. In
some embodiments, the subpopulation of cells having the largest
mean diameter within the plurality of input PBMCs is a population
of monocytes, wherein the diameter of the population of monocytes
is about 15 .mu.m to about 25 .mu.m. In some embodiments, the mean
diameter of the population of monocytes is about 20 .mu.m. In some
embodiments, the subpopulation of cells having the largest mean
diameter within the plurality of input PBMCs are monocytes.
[0322] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
diameter of the constriction is about 3 .mu.m to about 15 .mu.m. In
some embodiments, the diameter of the constriction is about 3 .mu.m
to about 10 .mu.m. In some embodiments, the diameter of the
constriction is about 3 .mu.m to about 6 .mu.m. In some
embodiments, the diameter of the constriction is about 4 .mu.m to
about 10 .mu.m. In some embodiments, the diameter of the
constriction is about 4.2 .mu.m to about 6 .mu.m. In some
embodiments, the diameter of the constriction is about 4.2 .mu.m to
about 4.8 .mu.m. In some embodiments, the diameter of the
constriction is any one of about 2 .mu.m to about 14 .mu.m, about 4
.mu.m to about 12 .mu.m, about 6 .mu.m to about 9 .mu.m, about 4
.mu.m to about 6 .mu.m, about 4 .mu.m to about 5 .mu.m, about 3.5
.mu.m to about 7 .mu.m, about 3.5 .mu.m to about 6.3 .mu.m, about
3.5 .mu.m to about 5.6 .mu.m, about 3.5 .mu.m to about 4.9 .mu.m,
about 4.2 .mu.m to about 6.3 .mu.m, about 4.2 .mu.m to about 5.6
.mu.m, or about 4.2 .mu.m to about 4.9 .mu.m. In some embodiments,
the diameter of the constriction is any one of about 2 .mu.m, 2.5
.mu.m, 3 .mu.m, 3.5 .mu.m, 4 .mu.m, 4.5 .mu.m, 5 .mu.m, 5.5 .mu.m,
6 .mu.m, 6.5 .mu.m, 7 .mu.m, 7.5 .mu.m, 8 .mu.m, 8.5 .mu.m, 9
.mu.m, 9.5 .mu.m, 10 .mu.m, 10.5 .mu.m, 11 .mu.m, 11.5 .mu.m, 12
.mu.m, 12.5 .mu.m, 13 .mu.m, 13.5 .mu.m, 14 .mu.m, 14.5 .mu.m or 15
.mu.m. In some embodiments, the diameter of the constriction is any
one of about 4.0 .mu.m, 4.1 .mu.m, 4.2 .mu.m, 4.3 .mu.m, 4.4 .mu.m,
4.5 .mu.m, 4.6 .mu.m, 4.7 .mu.m, 4.8 .mu.m, 4.9 .mu.m, or 5.0
.mu.m. In some embodiments, the diameter of the constriction is
about 4.5 .mu.m.
[0323] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of input PBMCs is passed through the constriction under a
pressure ranging from about 20 psi to about 150 psi. In some
embodiments, the plurality of input PBMCs is passed through the
constriction under a pressure ranging from about 30 psi to about
120 psi. In some embodiments, the plurality of input PBMCs is
passed through the constriction under a pressure ranging from about
60 psi to about 90 psi. In some embodiments, the plurality of input
PBMCs is passed through the constriction under a pressure ranging
from any one of about 30 psi to about 40 psi, about 40 psi to about
50 psi, about 50 psi to about 60 psi, about 60 psi to about 70 psi,
about 70 psi to about 80 psi, about 80 psi to about 90 psi, about
90 psi to about 100 psi, about 100 psi to about 110 psi, or about
110 psi to about 120 psi. In some embodiments, the plurality of
input PBMCs is passed through the constriction under a pressure of
about any one of 20 psi, 25 psi, 30 psi, 35 psi, 40 psi, 45 psi, 50
psi, 55 psi, 60 psi, 65 psi, 70 psi, 75 psi, 80 psi, 85 psi, 90
psi, 95 psi, 100 psi, 105 psi, 110 psi, 115 psi, or 120 psi.
[0324] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of input PBMCs is passed through the constriction under a
pressure ranging from about 150 kPa to about 1000 kPa. In some
embodiments, the plurality of input PBMCs is passed through the
constriction under a pressure ranging from about 207 kPa to about
830 kPa. In some embodiments, the plurality of input PBMCs is
passed through the constriction under a pressure ranging from about
415 kPa to about 621 kPa. In some embodiments, the plurality of
input PBMCs is passed through the constriction under a pressure
ranging from any one of about 200 kPa to about 250 kPa, about 250
kPa to about 300 kPa, 300 kPa to about 350 kPa, about 350 kPa to
about 400 kPa, 400 kPa to about 450 kPa, about 450 kPa to about 500
kPa, 500 kPa to about 550 kPa, about 550 kPa to about 600 kPa, 600
kPa to about 650 kPa, about 650 kPa to about 700 kPa, 700 kPa to
about 750 kPa, about 750 kPa to about 800 kPa, 800 kPa to about 850
kPa, about 850 kPa to about 900 kPa, 900 kPa to about 950 kPa,
about 950 kPa to about 1000 kPa. In some embodiments, the plurality
of input PBMCs is passed through the constriction under a pressure
of about any one of 200 kPa, 250 kPa, 300 kPa, 350 kPa, 400 kPa,
415 kPa, 450 kPa, 500 kPa, 550 kPa, 600 kPa, 612 kPa, 650 kPa, 700
kPa, 750 kPa, 800 kPa, or 850 kPa.
[0325] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of input PBMCs is passed through the constriction at a
temperature ranging from about 0.degree. C. to about 37.degree. C.
In some embodiments the plurality of input PBMCs is passed through
the constriction at a temperature ranging from about 0.degree. C.
to about 10.degree. C. In some embodiments, the plurality of input
PBMCs is passed through the constriction at a temperature ranging
from about 2.degree. C. to about 8.degree. C. In some embodiments,
the plurality of input PBMCs is passed through the constriction at
a temperature ranging from any one of about 2.degree. C. to about
6.degree. C., about 5.degree. C. to about 10.degree. C., about
10.degree. C. to about 15.degree. C., about 15.degree. C. to about
20.degree. C., about 20.degree. C. to about 25.degree. C., about
25.degree. C. to about 30.degree. C., about 30.degree. C. to about
35.degree. C., or about 35.degree. C. to about 37.degree. C. In
some embodiments, the plurality of input PBMCs is passed through
the constriction at a temperature of any one of about 0.degree. C.,
1.degree. C., 2.degree. C., 3.degree. C., 4.degree. C., 5.degree.
C., 6.degree. C., 7.degree. C., 8.degree. C., 9.degree. C.,
10.degree. C., 15.degree. C., 20.degree. C., 25.degree. C.,
30.degree. C. or 37.degree. C.
[0326] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein,
subsequent to passing through the constriction the plurality of
modified PBMCs is incubated at a temperature of 37.degree. C. for a
sufficient time to allow the modified PBMCs to normalize to
37.degree. C. In some embodiments, subsequent to passing through
the constriction the plurality of modified PBMCs is incubated at a
temperature of 25.degree. C. for a sufficient time to allow the
modified PBMCs to normalize to 25.degree. C.
[0327] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
input PBMCs is passed through the constriction at a flow rate
between about 0.001 mL/min to about 200 mL/min or any rate or range
of rates therebetween. In some embodiments, the flow rate is
between about 0.001 mL/min to about 175 mL/min, about 0.001 mL/min
to about 150 mL/min, about 0.001 mL/min to about 125 mL/min, about
0.001 mL/min to about 100 mL/min, about 0.001 mL/min to about 50
mL/min, about 0.001 mL/min to about 25 mL/min, about 0.001 mL/min
to about 10 mL/min, about 0.001 mL/min to about 7.5 mL/min, about
0.001 mL/min to about 5.0 mL/min, about 0.001 mL/min to about 2.5
mL/min, about 0.001 mL/min to about 1 mL/min, about 0.001 mL/min to
about 0.1 mL/min or about 0.001 mL/min to about 0.01 mL/min. In
some embodiments, the flow rate is between about 0.001 mL/min to
about 200 mL/min, about 0.01 mL/min to about 200 mL/min, about 0.1
mL/min to about 200 mL/min, about 1 mL/min to about 200 mL/min,
about 10 mL/min to about 200 mL/min, about 50 mL/min to about 200
mL/min, about 75 mL/min to about 200 mL/min, about 100 mL/min to
about 200 mL/min, about 150 mL/min to about 200 mL/min, about 0.5
mL/min to about 200 mL/min, about 1 mL/min to about 200 mL/min,
about 2.5 mL/min to about 200 mL/min, about 5 mL/min to about 200
mL/min, about 7.5 mL/min to about 200 mL/min, about 10 mL/min to
about 200 mL/min, about 25 mL/min to about 200 mL/min, or about 175
mL/min to about 200 mL/min. In some embodiments, the plurality of
input PBMCs is passed through the constriction at a flow rate
between about 10 mL/min to about 200 mL/min. In some embodiments,
the plurality of input PBMCs is passed through the constriction at
a flow rate of about 100 mL/min.
[0328] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
constriction can have any shape known in the art; e.g. a
3-dimensional shape or a 2-dimensional shape. The 2-dimensional
shape, such as the cross-sectional shape, of the constriction can
be, without limitation, circular, elliptical, round, square,
star-shaped, triangular, polygonal, pentagonal, hexagonal,
heptagonal, or octagonal. The 3-dimensional shape of the
constriction can be, without limitation, cylindrical, conical, or
cuboidal. In some embodiments, the cross-sectional shape of the
constriction is a rectangle. In some embodiments, the
cross-sectional shape of the constriction is a slit. In some
embodiments, the cross-sectional shape of the constriction is a
slit comprising a width of about 3 .mu.m to about 10 .mu.m and/or a
depth of about 1 .mu.m to about 200 .mu.m. In some embodiments, the
cross-sectional shape of the constriction is a slit comprising a
width of about 3 .mu.m to about 6 .mu.m and/or a depth of about 20
.mu.m to about 120 .mu.m. In some embodiments, the cross-sectional
shape of the constriction is a slit comprising a width of about 4.2
.mu.m to about 6 .mu.m and/or a depth of about 20 .mu.m to about
120 .mu.m. In some embodiments, the cross-sectional shape of the
constriction is a slit comprising a width of about 4.2 .mu.m to
about 6 .mu.m and/or a depth of about 40 .mu.m to about 100 .mu.m.
In some embodiments, the cross-sectional shape of the constriction
is a slit comprising a width of about 4.2 .mu.m to about 6 .mu.m
and/or a depth of about 20 .mu.m to about 80 .mu.m. In some
embodiments, the cross-sectional shape of the constriction is a
slit comprising a width of about 4.5 .mu.m and/or a depth of about
80 .mu.m. In some embodiments, the slit comprises a length of about
10 .mu.m to about 30 .mu.m. In some embodiments, the slit comprises
a length of about 2 .mu.m to about 50 .mu.m. In some embodiments,
the slit comprises a length of any one of about 2 .mu.m to about 5
.mu.m, about 5 .mu.m to about 10 .mu.m, about 10 .mu.m to about 15
.mu.m, about 15 .mu.m to about 20 .mu.m, about 20 .mu.m to about 25
.mu.m, about 25 .mu.m to about 30 .mu.m, about 30 .mu.m to about 35
.mu.m, about 35 .mu.m to about 40 .mu.m, about 40 .mu.m to about 45
.mu.m, or about 45 .mu.m to about 50 .mu.m. In some embodiments,
the slit comprises a length of about 10 .mu.m.
[0329] In some embodiments, the constriction comprises an entrance
portion and an exit portion. The entrances and exits of the
constriction may have a variety of angles. In some embodiments, the
constrictions have identical entrance and exit angles. In some
embodiments, the constrictions have different entrance and exit
angles. The constriction angle can be selected to minimize clogging
of the constriction while PBMCs are passing through. In some
embodiments the flow rate through the surface is between about
0.001 mL/min to about 100 mL/min or any rate or range of rates
therebetween. In some examples, the angle of the entrance and/or
exit portion can be between about 0 and about 90 degrees. In some
embodiments, the entrance and/or exit portion can be greater than
90 degrees. In some embodiments, the entrance portion defines an
entrance angle and the entrance angle is between about 0 degree to
about 90 degrees. In some embodiments, the entrance angle is
between any one of about 10 degrees to about 40 degrees, about 12
degrees to about 45 degrees, between about 15 degrees to about 30
degrees. In some embodiments, the entrance angle is between about
20 degrees to about 22 degrees. In some embodiments, the exit
portion defines an exit angle and the exit angle is between about 0
degree to about 90 degrees. In some embodiments, the exit angle is
between any one of about 10 degrees to about 40 degrees, about 12
degrees to about 45 degrees, between about 15 degrees to about 30
degrees. In some embodiments, the exit angle is between about 20
degrees to about 22 degrees. In some embodiments, the entrance
portion defines an entrance angle and the entrance angle is between
about 20 degrees to about 22 degrees, and the exit portion defines
an exit angle and the exit angle is between about 20 degrees to
about 22 degrees.
[0330] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
constriction edge is smooth, e.g. rounded or curved. A smooth
constriction edge has a continuous, flat, and even surface without
bumps, ridges, or uneven parts. In some embodiments, the
constriction edge is sharp. A sharp constriction edge has a thin
edge that is pointed or at an acute angle. In some embodiments, the
constriction passage is straight. A straight constriction passage
does not contain curves, bends, angles, or other irregularities. In
some embodiments, the constriction passage is curved. A curved
constriction passage is bent or deviates from a straight line. In
some embodiments, the constriction passage has multiple curves,
e.g. about 2, 3, 4, 5, 6, 7, 8, 9, 10 or more curves.
[0331] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
cell suspension comprising the plurality of input PBMCs is passed
through multiple constrictions, wherein the multiple constrictions
are arranged in series and/or in parallel. In some embodiments, the
multiple constrictions are arranged in series. In some embodiments,
the multiple constrictions are arranged in parallel. In some
embodiments, the multiple constrictions are arranged in series
and/or in parallel. In some embodiments, the multiple constrictions
arranged in series comprise about any one of 2, 3, 4, 5, 6, 7, 8,
9, 10, 50, 75, 100, 500, 1,000 or more constrictions in series. In
some embodiments, the multiple constrictions arranged in parallel
may comprise about any one of 2, 5, 10, 50, 75, 100, 500, 1,000 or
more constrictions in series.
[0332] Exemplary microfluidic channels containing cell-deforming
constrictions for use in the methods disclosed herein are described
in WO2013059343. Exemplary surfaces having pores for use in the
methods disclosed herein are described in WO2017041050.
Systems and Kits
[0333] In some aspects, the invention provides a system comprising
one or more of a constriction, a PBMC suspension, antigens or
adjuvants according to any of the embodiments described herein,
such as for use in any of the methods described herein. The system
can include any embodiment described for the compositions of matter
and methods disclosed herein, including those disclosed in the
above section titled "Microfluidic systems and components thereof"
In some embodiment, the cell-deforming constrictions are sized for
delivery to PBMCs. In some embodiments, the delivery parameters,
such as operating flow speeds, cell and compound concentration,
temperature, velocity of the cell in the constriction, and the
composition of the cell suspension (e.g., osmolarity, salt
concentration, serum content, cell concentration, pH, etc.) are
optimized for maximum response of a compound for modulating an
immune response.
[0334] Also provided are kits or articles of manufacture for use in
modulating an immune response in an individual. In some
embodiments, the kit comprises modified PBMCs comprising an antigen
and/or an adjuvant, including any of the modified PBMCs described
herein. In some embodiments, the kit comprises one or more of a
constriction, a PBMC suspension, antigens or adjuvants for use in
generating modified PBMCs for use in modulating an immune response
in an individual. In some embodiments, the kits comprise components
described herein (e.g. a microfluidic channel or surface containing
pores, cell suspensions, and/or compounds) in suitable packaging.
Suitable packaging materials are known in the art, and include, for
example, vials (such as sealed vials), vessels, ampules, bottles,
jars, flexible packaging (e.g., sealed Mylar or plastic bags), and
the like. These articles of manufacture may further be sterilized
and/or sealed.
[0335] The invention also provides kits comprising components of
the methods described herein and may further comprise instructions
for performing said methods to modulate an immune response in an
individual and/or instructions for introducing an antigen and/or an
adjuvant into PBMCs. The kits described herein may further include
other materials, including buffers, diluents, filters, needles,
syringes, and package inserts with instructions for performing any
of the methods described herein; e.g., instructions for modulating
an immune response in an individual or instructions for modifying a
PBMCs to contain an antigen and/or an adjuvant. HPV and
HPV-associated diseases.
[0336] In some embodiments according to any one of systems and kits
described herein, the antigen comprises one or more proteins. In
some embodiments, the antigen is encoded by one or more nucleic
acids and enters the PBMC in the form of one or more nucleic acids,
such as but not limited to DNAs, cDNAs, mRNAs, and plasmids. In
some embodiments, the antigen is encoded by one or more mRNAs and
enters the PBMC in the form of one or more mRNAs.
Other Embodiments
[0337] Other embodiments provide any of the embodiments described
herein with one or more of the following provisos: [0338] the
antigen is not an HPV antigen [0339] the antigen is not an HPV E6
antigen [0340] the antigen is not an HPV E7 antigen [0341] the
antigen is not an HPV E6 antigen and not an HPV E7 antigen [0342]
an adjuvant is not introduced into the PBMCs together with the
antigen [0343] an adjuvant is not presented in the cytosol of the
PBMC comprising the antigen [0344] the adjuvant is not administered
to the individual
[0345] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of modified PBMCs does not express an HPV antigen. In
some embodiments, the plurality of modified PBMCs does not comprise
a nucleic acid encoding an HPV antigen. In some embodiments, the
plurality of modified PBMCs does not comprise an HPV E6 antigen. In
some embodiments, the plurality of modified PBMCs does not comprise
a nucleic acid encoding an HPV E6 antigen. In some embodiments, the
plurality of modified PBMCs does not comprise an HPV E7 antigen. In
some embodiments, the plurality of modified PBMCs does not comprise
a nucleic acid encoding an HPV E7 antigen.
[0346] In some embodiments, the plurality of modified PBMCs does
not comprise an HPV E6 antigen and does not comprise an HPV E7
antigen. In some embodiments, the plurality of modified PBMCs does
not comprise a nucleic acid encoding an HPV E6 antigen and does not
comprise a nucleic acid encoding an HPV E7 antigen.
[0347] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of PBMCs does comprise nucleic acid encoding an antigen.
In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of PBMCs do not express an antigen.
[0348] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
antigen comprises one or more proteins. In some embodiments, the
antigen is encoded by one or more nucleic acids and enters the PBMC
in the form of one or more nucleic acids, such as but not limited
to DNAs, cDNAs, mRNAs, and plasmids. In some embodiments, the
antigen is encoded by one or more mRNAs and enters the PBMC in the
form of one or more mRNAs. In some embodiments, the plurality of
PBMCs comprises a nucleic acid encoding an antigen. In some
embodiments, the plurality of PBMCs comprises an mRNA encoding an
antigen.
[0349] In some embodiments according to any one of the methods,
compositions or pluralities of modified PBMCs described herein, the
plurality of PBMCs does not induce tolerance in an individual. In
some embodiments, the plurality of PBMCs does not suppress an
immune response in an individual. In some embodiments, the
plurality of PBMCs does not comprise a tolerogenic factor. In some
embodiments, the plurality of PBMCs is not administered in
combination with a tolerogenic factor. In some embodiments, the
plurality of PBMCs is not administered before, simultaneous with,
or after administration of a tolerogenic factor.
[0350] In some embodiments of the application, the terms
"conditioned" and "matured" may be used interchangeably.
EXEMPLARY EMBODIMENTS
[0351] The invention provides the following enumerated
embodiments.
[0352] 1. A plurality of modified PBMCs comprising an antigen,
wherein the antigen is exogenous to the modified PBMCs.
[0353] 2. A plurality of modified PMBCs comprising an antigen,
wherein the antigen is exogenous to the modified PBMCs, wherein the
antigen is a cancer antigen, an infectious disease antigen or a
viral-disease associated antigen.
[0354] 3. A conditioned plurality of modified PBMCs comprising an
antigen, wherein the antigen is exogenous to the modified
PBMCs.
[0355] 4. A conditioned plurality of modified PMBCs comprising an
antigen, wherein the antigen is exogenous to the modified PBMCs,
wherein the antigen is a cancer antigen, an infectious disease
antigen or a viral-disease associated antigen.
[0356] 5. A conditioned plurality of modified PBMCs comprising an
antigen and an adjuvant, wherein the antigen is exogenous to the
modified PBMCs.
[0357] 6. A plurality of modified PBMCs comprising an antigen
comprising the amino acid sequence of any one of SEQ ID NOs:
18-25.
[0358] 7. A conditioned plurality of modified PBMCs comprising an
antigen comprising the amino acid sequence of any one of SEQ ID
NOs: 18-25.
[0359] 8. A conditioned plurality of PBMCs comprising an antigen,
prepared by incubating the plurality of PBMCs comprising the
antigen with an adjuvant for a sufficient time for the PBMCs to
condition, thereby generating the conditioned plurality of PBMCs
comprising the antigen.
[0360] 9. A conditioned plurality of PBMCs comprising an antigen,
prepared by incubating the plurality of PBMCs with an adjuvant for
a sufficient time for the PBMCs to condition prior to introducing
the antigen to the PBMCs, thereby generating the conditioned
plurality of PBMCs comprising the antigen.
[0361] 10. A plurality of modified PBMCs comprising an antigen,
prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen.
[0362] 11. A plurality of modified PBMCs comprising an antigen,
prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, wherein
the nucleic acid is expressed in the PBMCs to produce the antigen,
thereby generating a plurality of modified PBMCs comprising the
antigen.
[0363] 12. A conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0364] 13. A conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; and c) incubating the plurality of
modified PBMCs with the nucleic acid encoding the antigen with an
adjuvant for a sufficient time for the modified PBMCs comprising
the nucleic acid encoding the antigen to condition, wherein the
nucleic acid is expressed in the PBMCs to produce the antigen,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen.
[0365] 14. The conditioned plurality of modified PBMCs comprising
an antigen of embodiment 12 or 13, wherein the process further
comprises: isolating the plurality of modified PBMCs comprising the
antigen from the cell suspension before incubation with the
adjuvant to condition the modified PBMCs.
[0366] 15. A plurality of modified PBMCs comprising an antigen and
an adjuvant, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen and the adjuvant to pass through to form a
plurality of perturbed input PBMCs; and b) incubating the plurality
of perturbed input PBMCs with the antigen and the adjuvant for a
sufficient time to allow the antigen and the adjuvant to enter the
perturbed input PBMCs; thereby generating the plurality of modified
PBMCs comprising the antigen and adjuvant.
[0367] 16. A plurality of modified PBMCs comprising an antigen and
an adjuvant, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen and the adjuvant to
pass through to form a plurality of perturbed input PBMCs; and b)
incubating the plurality of perturbed input PBMCs with the nucleic
acid encoding the antigen and with the adjuvant for a sufficient
time to allow the nucleic acid encoding the antigen and the
adjuvant to enter the perturbed input PBMCs; wherein the nucleic
acid is expressed in the PBMCs to produce the antigen, thereby
generating the plurality of modified PBMCs comprising the antigen
and adjuvant.
[0368] 17 The plurality of modified PBMCs of embodiment 15 or 16,
wherein the concentration of the antigen incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 1 mM
and/or the concentration of the adjuvant incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 1
mM.
[0369] 18. The plurality of modified PBMCs of any one of
embodiments 15-17, wherein: (a) the concentration of the antigen
incubated with the perturbed input PBMCs is between about 0.1 .mu.M
and about 10 .mu.M and/or the concentration of the adjuvant
incubated with the perturbed input PBMCs is between about 0.1 .mu.M
and about 10 .mu.M.
[0370] 19. The plurality of modified PBMCs of any one of
embodiments 15-18, wherein the concentration of the antigen
incubated with the perturbed input PBMCs is about 1 .mu.M and/or
the concentration of the adjuvant incubated with the perturbed
input PBMCs is about 1 .mu.M.
[0371] 20. The plurality of modified PBMCs of any one of
embodiments 15-19, wherein the ratio of the antigen to the adjuvant
incubated with the perturbed input PBMCs is between about 10000:1
to about 1:10000.
[0372] 21. The plurality of modified PBMCs of any one of
embodiments 15-20, wherein the ratio of the antigen to the adjuvant
incubated with the perturbed input PBMCs is about 200:1.
[0373] 22. A conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; and c) incubating the
conditioned plurality of perturbed input PBMCs with the antigen for
a sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0374] 23. The plurality of modified PBMCs of embodiment 22,
wherein the concentration of the adjuvant incubated with the input
PBMCs is between about 0.1 .mu.M and about 1 mM.
[0375] 24. The plurality of modified PBMCs of embodiment 22 or 23,
wherein the concentration of the adjuvant incubated with the input
PBMCs is between about 0.1 .mu.M and about 10 .mu.M.
[0376] 25. The plurality of modified PBMCs of any one of
embodiments 22-24, wherein the concentration of the adjuvant
incubated with the input PBMCs is about 1 .mu.M.
[0377] 26. A plurality of modified PBMCs comprising an antigen and
an adjuvant, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
comprising the adjuvant through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the antigen to pass through to
form a plurality of perturbed input PBMCs; and b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen and the adjuvant.
[0378] 27. A plurality of modified PBMCs comprising an antigen and
an adjuvant, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
comprising the antigen through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for the adjuvant to pass through to
form a plurality of perturbed input PBMCs; and b) incubating the
plurality of perturbed input PBMCs with the adjuvant for a
sufficient time to allow the adjuvant to enter the perturbed input
PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen and the adjuvant.
[0379] 28. The plurality of modified PBMCs of any one of
embodiments 10-14, and 22-27, wherein the concentration of the
antigen incubated with the perturbed input PBMCs is between about
0.1 .mu.M and about 1 mM.
[0380] 29. The plurality of modified PBMCs of any one of
embodiments 10-14, and 22-28, wherein the concentration of the
antigen incubated with the perturbed input PBMCs is between about
0.1 .mu.M and about 10 .mu.M.
[0381] 30. The plurality of modified PBMCs of any one of
embodiments 10-14, and 22-29 wherein the concentration of the
antigen incubated with the perturbed input PBMCs is about 1
.mu.M.
[0382] 31. The plurality of modified PBMCs comprising the antigen
and/or the adjuvant according to any one of embodiments 15-21, and
26-30, wherein the process further comprises: incubating the
plurality of modified PBMCs comprising the antigen and/or adjuvant
with a second adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen
and/or the adjuvant.
[0383] 32. The plurality of modified PBMCs comprising an antigen
and/or the adjuvant of embodiment 31, wherein the process further
comprises: isolating the plurality of modified PBMCs comprising the
antigen and/or the adjuvant from the cell suspension before
incubation with the adjuvant to condition the modified PBMCs.
[0384] 33. The plurality of modified PBMCs of any one of
embodiments 8-31, wherein the concentration of the adjuvant
incubated with the modified PBMCs is between about 0.1 .mu.M and
about 1 mM.
[0385] 34. The plurality of modified PBMCs of any one of
embodiments 8-33, wherein the concentration of the adjuvant
incubated with the modified PBMCs is between about 0.1 .mu.M and
about 10 .mu.M.
[0386] 35. The plurality of modified PBMCs of any one of
embodiments 8-34, wherein the concentration of the adjuvant
incubated with the modified PBMCs is about 1 .mu.M.
[0387] 36. The plurality of modified PBMCs of any one of
embodiments 8-35, wherein the process further comprises a step of
incubating the input PBMCs and/or the modified PBMCs with an agent
that enhances the viability and/or function of the modified PBMCs
as compared to corresponding modified PBMCs prepared without the
further incubation step.
[0388] 37. The plurality of modified PBMCs of any one of
embodiments 10-36, wherein the diameter of the constriction is
about 10% to about 99% of the mean diameter of the plurality of
input PBMCs.
[0389] 38. The plurality of modified PBMCs of any one of
embodiments 10-37, wherein the diameter of the constriction is
about 10% to about 70% of the mean diameter of the plurality of
input PBMCs.
[0390] 39. The plurality of modified PBMCs of any one of
embodiments 10-38, wherein the diameter of the constriction is
about 20% to about 60% of the mean diameter of the plurality of
input PBMCs.
[0391] 40. The plurality of modified PBMCs of any one of
embodiments 10-39, wherein the diameter of the constriction is
about 40% to about 60% of the mean diameter of the plurality of
input PBMCs.
[0392] 41. The plurality of modified PBMCs of any one of
embodiments 10-40, wherein the diameter of the constriction is
about 30% to about 45% of the mean diameter of the plurality of
input PBMCs.
[0393] 42. The plurality of modified PBMCs of any one of
embodiments 10-36, wherein the diameter of the constriction is
about 10% to about 99% of the mean diameter of a subpopulation of
cells having the smallest diameter within the plurality of input
PBMCs.
[0394] 43. The plurality of modified PBMCs of any one of
embodiments 10-36 and 42, wherein the diameter of the constriction
is about 10% to about 70% of the mean diameter of a subpopulation
of cells having the smallest diameter within the plurality of input
PBMCs.
[0395] 44. The plurality of modified PBMCs of any one of
embodiments 10-36, 42 and 43 wherein the diameter of the
constriction is about 20% to about 60% of the mean diameter of a
subpopulation of cells having the smallest diameter within the
plurality of input PBMCs.
[0396] 45. The plurality of modified PBMCs of any one of
embodiments 10-36 and 42-44 wherein the diameter of the
constriction is about 30% to about 45% of the mean diameter of a
subpopulation of cells having the smallest diameter within the
plurality of input PBMCs.
[0397] 46. The plurality of modified PBMCs of any one of
embodiments 10-36, wherein the diameter of the constriction is
about 50% to about 99% of the mean diameter of a subpopulation of
cells having the smallest diameter within the plurality of input
PBMCs.
[0398] 47. The plurality of modified PBMCs of any one of
embodiments 10-36 and 46, wherein the diameter of the constriction
is about 50% to about 90% of the mean diameter of a subpopulation
of cells having the smallest diameter within the plurality of input
PBMCs.
[0399] 48. The plurality of modified PBMCs of any one of
embodiments 10-36, 46 and 47, wherein the diameter of the
constriction is about 50% to about 80% of the mean diameter of a
subpopulation of cells having the smallest diameter within the
plurality of input PBMCs.
[0400] 49. The plurality of modified PBMCs of any one of
embodiments 10-36 and 46-48, wherein the diameter of the
constriction is about 50% to about 70% of the mean diameter of a
subpopulation of cells having the smallest diameter within the
plurality of input PBMCs.
[0401] 50. The plurality of modified PBMCs of any one of
embodiments 10-36, wherein the diameter of the constriction is
about 60% to about 90% of the mean diameter of a subpopulation of
cells having the smallest diameter within the plurality of input
PBMCs.
[0402] 51. The plurality of modified PBMCs of any one of
embodiments 10-36 and 50, wherein the diameter of the constriction
is about 60% to about 80% of the mean diameter of a subpopulation
of cells having the smallest diameter within the plurality of input
PBMCs.
[0403] 52. The plurality of modified PBMCs of any one of
embodiments 10-36, 50 and 51, wherein the diameter of the
constriction is about 60% to about 70% of the mean diameter of a
subpopulation of cells having the smallest diameter within the
plurality of input PBMCs.
[0404] 53. The plurality of modified PBMCs of any one of
embodiments 10-52, wherein the diameter of the constriction is
about 10% to about 99% of the mean diameter of a subpopulation of
cells having the largest diameter within the plurality of input
PBMCs.
[0405] 54. The plurality of modified PBMCs of any one of
embodiments 10-53, wherein the diameter of the constriction is
about 10% to about 70% of the mean diameter of a subpopulation of
cells having the largest diameter within the plurality of input
PBMCs.
[0406] 55. The plurality of modified PBMCs of any one of
embodiments 10-54 wherein the diameter of the constriction is about
20% to about 60% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0407] 56. The plurality of modified PBMCs of any one of
embodiments 10-55 wherein the diameter of the constriction is about
20% to about 30% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0408] 57. The plurality of modified PBMCs of any one of
embodiments 10-56 wherein the diameter of the constriction is about
20% to about 25% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0409] 58. The plurality of modified PBMCs of any one of
embodiments 10-55 wherein the diameter of the constriction is about
30% to about 70% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0410] 59. The plurality of modified PBMCs of any one of
embodiments 10-55 and 58 wherein the diameter of the constriction
is about 30% to about 60% of the mean diameter of a subpopulation
of cells having the largest diameter within the plurality of input
PBMCs.
[0411] 60. The plurality of modified PBMCs of any one of
embodiments 10-55, 58 and 59 wherein the diameter of the
constriction is about 30% to about 45% of the mean diameter of a
subpopulation of cells having the largest diameter within the
plurality of input PBMCs.
[0412] 61. The plurality of modified PBMCs of any one of
embodiments 42-60, wherein the subpopulation of cells having the
smallest diameter within the plurality of input PBMCs are T
cells.
[0413] 62. The plurality of modified PBMCs of any one of
embodiments 53-61, wherein the subpopulation of cells having the
largest diameter within the plurality of input PBMCs are
monocytes.
[0414] 63. The plurality of modified PBMCs of any one of
embodiments 10-62, wherein the diameter of the constriction is
about 3 .mu.m to about 10 .mu.m.
[0415] 64. The plurality of modified PBMCs of any one of
embodiments 10-63, wherein the diameter of the constriction is
about 4 .mu.m to about 10 .mu.m.
[0416] 65. The plurality of modified PBMCs of any one of
embodiments 10-63, wherein the diameter of the constriction is
about 3 .mu.m to about 6 .mu.m.
[0417] 66. The plurality of modified PBMCs of any one of
embodiments 10-65, wherein the diameter of the constriction is
about 4.2 .mu.m to about 6 .mu.m.
[0418] 67. The plurality of modified PBMCs of any one of
embodiments 10-66, wherein the diameter of the constriction is
about 4.5 .mu.m.
[0419] 68. The plurality of modified PBMCs of any one of
embodiments 10-67, wherein the plurality of input PBMCs is passed
through the constriction under a pressure ranging from about 30 psi
to about 120 psi or about 60 psi to about 90 psi.
[0420] 69. The plurality of modified PBMCs of any one of
embodiments 10-67, wherein the plurality of input PBMCs is passed
through the constriction under a pressure ranging from about 207
kPa to about 830 kPa or about 415 kPa to about 621 kPa.
[0421] 70. The plurality of modified PBMCs of any one of
embodiments 10-67, wherein the plurality of input PBMCs is passed
through the constriction at a flow rate between about 0.001
mL/cm.sup.2/sec to about 200 L/cm.sup.2/sec.
[0422] 71. The plurality of modified PBMCs of any one of
embodiments 10-67, wherein the plurality of input PBMCs is passed
through the constriction at a flow rate between about 0.1
mL/cm.sup.2/sec to about 150 L/cm.sup.2/sec.
[0423] 72. The plurality of modified PBMCs of any one of
embodiments 10-67, wherein the plurality of input PBMCs is passed
through the constriction at a flow rate of about 100
mL/cm.sup.2/sec.
[0424] 73. The plurality of modified PBMCs of any one of
embodiments 10-72, wherein the plurality of input PBMCs is passed
through the constriction at a temperature ranging from about
0.degree. C. to about 37.degree. C.
[0425] 74. The plurality of modified PBMCs of any one of
embodiments 10-73, wherein subsequent to passing through the
constriction the plurality of modified PBMCs is incubated at a
temperature of 37.degree. C. for a sufficient time to allow the
modified PBMCs to normalize to 37.degree. C.
[0426] 75. The plurality of modified PBMCs of any one of
embodiments 10-74, wherein subsequent to passing through the
constriction the plurality of modified PBMCs is incubated at a
temperature of 25.degree. C. for a sufficient time to allow the
modified PBMCs to normalize to 25.degree. C.
[0427] 76. The plurality of modified PBMCs of any one of
embodiments 10-75, wherein the cross-sectional shape of the
constriction is selected from the group consisting of: circular,
elliptical, round, square, rectangular, star-shaped, triangular,
polygonal, pentagonal, hexagonal, heptagonal, and octagonal.
[0428] 77. The plurality of modified PBMCs of any one of
embodiments 10-76, wherein the cross-sectional shape of the
constriction is a slit.
[0429] 78. The plurality of modified PBMCs embodiment 77, wherein
slit comprises a width of about 3 .mu.m-6 .mu.m and/or a depth of
about 20 .mu.m-120 .mu.m.
[0430] 79. The plurality of modified PBMCs embodiment 78, wherein
the slit comprises a width of about 4.2 .mu.m-6 .mu.m and/or a
depth of about 20 .mu.m-120 .mu.m.
[0431] 80. The plurality of modified PBMCs of any one of
embodiments 77-79, wherein the slit comprises a width of about 4.5
.mu.m and/or a depth of about 80 .mu.m.
[0432] 81. The plurality of modified PBMCs of any one of
embodiments 10-80, wherein the cell suspension comprising the
plurality of input PBMCs are passed through multiple constrictions
wherein the multiple constrictions are arranged in series and/or in
parallel.
[0433] 82. The plurality of modified PBMCs of any one of
embodiments 10-80, wherein the constriction comprises an entrance
portion and an exit portion, wherein:
(a) the entrance portion defines an entrance angle and the entrance
angle is between about 0 degree to about 90 degrees or between
about 20-22 degrees; and/or (b) the exit portion defines an exit
angle and the exit angle is between about 0 degree to about 90
degrees or between about 20-22 degrees; preferably between about
20-22 degrees for (a) and (b).
[0434] 83. The plurality of modified PBMCs of any one of
embodiments 10-82, wherein the cell suspension comprising the
plurality of input PBMCs are passed through multiple constrictions
wherein the multiple constrictions are arranged in series and/or in
parallel.
[0435] 84. The conditioned plurality of modified PBMCs of any one
of embodiments 12-14, 22-25, 31-83, wherein the plurality of
modified PBMCs is incubated with the adjuvant for about 1 to about
24 hours for the modified PBMCs to condition.
[0436] 85. The conditioned plurality of modified PBMCs of any one
of embodiments 12-14, 22-25, 31-84, wherein the plurality of
modified PBMCs is incubated with the adjuvant for about 2 to about
10 hours for the modified PBMCs to condition.
[0437] 86. The conditioned plurality of modified PBMCs of any one
of embodiments 12-14, 22-25, 31-85, wherein the plurality of
modified PBMCs is incubated with the adjuvant for about 3 to about
6 hours for the modified PBMCs to condition.
[0438] 87. The conditioned plurality of modified PBMCs of any one
of embodiments 12-14, 22-25, 31-86, wherein the plurality of
modified PBMCs is incubated with the adjuvant for about 4 hours for
the modified PBMCs to condition.
[0439] 88. The plurality of modified PBMCs of any one of
embodiments 1-87, wherein the antigen, the nucleic acid encoding
the antigen, and/or adjuvant are present in the cytosol and/or a
vesicle of a cell in the plurality of modified PBMCs.
[0440] 89. The plurality of modified PBMCs of any one of
embodiments 1-88, wherein the antigen and/or the nucleic acid
encoding the antigen is present in the cytosol and the adjuvant is
present in a vesicle of a cell in the plurality of modified
PBMCs.
[0441] 90. The plurality of modified PBMCs of embodiment 88 or 89,
wherein the vesicle is an endosome.
[0442] 91. The plurality of modified PBMCs of any one of
embodiments 1-90, wherein the antigen, the nucleic acid encoding
the antigen, and/or the adjuvant are present in multiple
compartments of a cell in the plurality of modified PBMCs.
[0443] 92. The plurality of modified PBMCs of any one of
embodiments 1-91, wherein the antigen, the nucleic acid encoding
the antigen, and/or the adjuvant are present in at least about 70%
of the cells in the plurality of PBMCs.
[0444] 93. The plurality of modified PBMCs of any one of
embodiments 1-92, wherein the antigen, the nucleic acid encoding
the antigen, and/or the adjuvant are present in at least about 70%
of each of the T cells, B cells, NK cells, and monocytes in the
plurality of PBMCs.
[0445] 94. The plurality of modified PBMCs of any one of
embodiments 1-93, wherein the antigen is bound to the surface of a
cell in the plurality of modified PBMCs.
[0446] 95. The plurality of modified PBMCs of any one of
embodiments 5-94, wherein the adjuvant is a CpG
oligodeoxynucleotide (ODN), LPS, IFN-.alpha., STING agonists, RIG-I
agonists, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist or a
TLR 9 agonist.
[0447] 96. The plurality of modified PBMCs of embodiment 95,
wherein the adjuvant is a CpG ODN.
[0448] 97. The plurality of modified PBMCs of embodiment 96,
wherein the CpG ODN is a Class A CpG ODN, a Class B CpG ODN, or a
Class C CpG ODN.
[0449] 98. The plurality of modified PBMCs of any one of
embodiments 1-97, wherein the antigen is a disease-associated
antigen.
[0450] 99. The plurality of modified PBMCs of embodiment 98,
wherein the antigen is derived from peptides or mRNA isolated from
a diseased cell.
[0451] 100. The plurality of modified PBMCs of any one of
embodiments 1-99, wherein the antigen is a non-self antigen.
[0452] 101. The plurality of modified PBMCs of any one of
embodiments 1-100, wherein the antigen is a tumor antigen, viral
antigen, bacterial antigen, or fungal antigen.
[0453] 102. The plurality of modified PBMCs of any one of
embodiments 1-5 and 8-101, wherein the antigen is derived from a
tumor lysate.
[0454] 103. The plurality of modified PBMCs of any one of
embodiments 1-101, wherein the antigen is a human papillomavirus
(HPV) antigen.
[0455] 104. The plurality of modified PBMCs of embodiment 103,
wherein the HPV is HPV-16 or HPV-18.
[0456] 105. The plurality of modified PBMCs of embodiment 103 or
104, wherein the antigen comprises a peptide derived from HPV E6
and/or E7.
[0457] 106. The plurality of modified PBMCs of embodiment 103 or
104, wherein the antigen comprises an HLA-A2-restricted peptide
derived from HPV E6 and/or E7.
[0458] 107. The plurality of modified PBMCs of embodiment 106,
wherein the HLA-A2-restricted peptide comprises the amino acid
sequence of any one of SEQ ID NOs: 1-4.
[0459] 108. The plurality of modified PBMCs of embodiment 107,
wherein the antigen comprises the amino acid sequence of any one of
SEQ ID NOs: 18-25.
[0460] 109. The plurality of modified PBMCs of any one of
embodiments 1-108, wherein the modified PBMCs comprises a plurality
of antigens that comprise a plurality of immunogenic epitopes.
[0461] 110. The plurality of modified PBMCs of embodiment 109,
wherein following administration to an individual of the modified
PBMCs comprising the plurality of antigens that comprise the
plurality of immunogenic epitopes, none of the plurality of
immunogenic epitopes decreases an immune response in the individual
to any of the other immunogenic epitopes.
[0462] 111. The plurality of modified PBMCs of embodiment 110,
wherein the antigen is a polypeptide and the immunogenic epitope is
an immunogenic peptide epitope.
[0463] 112. The plurality of modified PBMCs of embodiment 111,
wherein the immunogenic peptide epitope is fused to an N-terminal
flanking polypeptide and/or a C-terminal flanking polypeptide.
[0464] 113. The plurality of modified PBMCs of embodiment 111,
wherein the antigen is a polypeptide comprising an immunogenic
peptide epitope and one or more heterologous peptide sequences.
[0465] 114. The plurality of modified PBMCs of embodiment 111,
wherein the antigen is a polypeptide comprising an immunogenic
peptide epitope that is flanked on the N-terminus and/or the
C-terminus by heterologous peptide sequences
[0466] 115. The plurality of modified PBMCs of embodiment 114,
wherein the flanking heterologous peptide sequences are derived
from a disease-associated immunogenic peptide.
[0467] 116. The plurality of modified PBMCs of embodiment 112,
wherein the N-terminal flanking polypeptide comprises the amino
acid sequence of any one of SEQ ID NOs: 5-10 and/or the C-terminal
flanking polypeptide comprises the amino acid sequence of any one
of SEQ ID NOs: 11-17.
[0468] 117. The plurality of modified PBMCs of any one of
embodiments 1-116, wherein the antigen is capable of being
processed into an MHC class I-restricted peptide and/or an MHC
class II-restricted peptide.
[0469] 118. The plurality of modified PBMCs of any one of
embodiments 5, 15-21 and 26-117, wherein the modified PBMCs
comprise the adjuvant at a concentration between about 1 nM and
about 1 mM.
[0470] 119. The plurality of modified PBMCs of any one of
embodiments 1-118, wherein the modified PBMCs comprise the antigen
at a concentration between about 1 nM and about 1 mM.
[0471] 120. The plurality of modified PBMCs of any one of
embodiments 1-119, wherein the ratio of the antigen to the adjuvant
is between about 10000:1 to about 1:10000.
[0472] 121. The plurality of modified PBMCs of any one of
embodiments 1-120, wherein the ratio of the antigen to the adjuvant
is about 200:1.
[0473] 122. The plurality of modified PBMCs of any one of
embodiments 1-118, wherein the modified PBMCs comprise a complex
comprising: a) the antigen, b) the antigen and at least one other
antigen, c) the antigen and the adjuvant, d) the nucleic acid
encoding the antigen, e) the nucleic acid encoding the antigen and
at least one other nucleic acid encoding one other antigen, and/or
f) the nucleic acid encoding the antigen and the adjuvant.
[0474] 123. The plurality of modified PBMCs of any one of
embodiments 3-5, 7-9, 12-14, 22-25 and 31-122, wherein the
plurality of modified PBMCs further comprises an agent that
enhances the viability and/or function of the plurality of modified
PBMCs as compared to a corresponding plurality of modified PBMCs
that does not comprise the agent.
[0475] 124. The plurality of modified PBMCs of any one of
embodiments 3-5, 7-9, 12-14, 22-25 and 31-123, wherein the
plurality of modified PBMCs further comprises an agent that
enhances the viability and/or function of the plurality of modified
PBMCs upon freeze-thaw cycle as compared to a corresponding
plurality of modified PBMCs that does not comprise the agent.
[0476] 125. The plurality of modified PBMCs of any one of
embodiments 3-5, 7-9, 12-14, 22-25 and 31-124, wherein at least
about 70%, about 80%, or about 90% of the conditioned plurality of
modified PBMCs are viable after up to 1, 2, 3, 4, 5 freeze-thaw
cycles.
[0477] 126. The plurality of modified PBMCs of any one of
embodiments 123-125, wherein the agent is a compound that enhances
endocytosis, a stabilizing agent or a co-factor.
[0478] 127. The plurality of modified PBMCs of any one of
embodiments 123-126, wherein the agent is albumin.
[0479] 128. The plurality of modified PBMCs of embodiment 127,
wherein the albumin is mouse, bovine, or human albumin.
[0480] 129. The plurality of modified PBMCs of any one of
embodiments 123-125, wherein the agent is one or more of: a
divalent metal cation, glucose, ATP, potassium, glycerol,
trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or
EDTA.
[0481] 130. The plurality of modified PBMCs of any one of
embodiments 123-125, wherein the agent is one or more of: Sodium
pyruvate, adenine, Rejuvesol.RTM., trehalose, dextrose, mannose,
sucrose, human serum albumin (HSA), PlasmaLyte.RTM., DMSO,
Cryostor.RTM. CS2, Cryostor.RTM. CS5, Cryostor.RTM. CS10,
Cryostor.RTM. CS15, HEPES, glycerol, glutathione,
HypoThermosol.RTM.
[0482] 131. The plurality of modified PBMCs of embodiment 128,
wherein the agent comprises mouse serum albumin (MSA).
[0483] 132. The plurality of modified PBMCs of embodiment 128,
wherein the agent comprises human serum albumin (HSA)
[0484] 133. The plurality of modified PBMCs of any one of
embodiments 1-132 wherein the cells are further modified to
increase expression of one or more of co-stimulatory molecules.
[0485] 134. The plurality of modified PBMCs of embodiment 133,
wherein the co-stimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80),
B7-2 (CD86), CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL,
CD30L, TIM4, SLAM, CD48, CD58, CD155, or CD112.
[0486] 135. The plurality of modified PBMCs of embodiment 133,
wherein the co-stimulatory molecule is a Signal 2 effector.
[0487] 136. The plurality of modified PBMCs of any one of
embodiments 133-135, wherein the cell comprises a nucleic acid
(e.g., mRNA) that results in increased expression of the one or
more co-stimulatory molecules.
[0488] 137. The plurality of modified PBMCs of embodiment 136,
wherein the nucleic acid encodes the costimulatory molecule.
[0489] 138. The plurality of modified PBMCs of any one of
embodiments 1-137 wherein the cells are further modified to
increase expression of one or more cytokines.
[0490] 139. The plurality of modified PBMCs of embodiment 138,
wherein the cytokine is IL-12, IL-2, IFN-.alpha., or IL-21.
[0491] 140. The plurality of modified PBMCs of embodiment 133,
wherein the co-stimulatory molecule is a Signal 3 effector.
[0492] 141. The plurality of modified PBMCs of any one of
embodiments 138-140, wherein the cell comprises a nucleic acid
(e.g., mRNA) that results in increased expression of the one or
more cytokines.
[0493] 142. The plurality of modified PBMCs of embodiment 141,
wherein the nucleic acid encodes the cytokine.
[0494] 143. The plurality of modified PBMCs of any one of
embodiments 1-142, wherein at least one cell in the plurality of
modified PBMCs is positive for expression of HLA-A2.
[0495] 144. The plurality of modified PBMCs of any one of
embodiments 1-142, wherein the modified PBMCs comprise a further
modification to modulate MHC class I expression.
[0496] 145. The plurality of modified PBMCs of any one of
embodiments 1-142, wherein the modified PBMCs comprise a further
modification to modulate HLA-A02 MHC I.
[0497] 146. The plurality of modified PBMCs of any one of
embodiments 1-145, wherein the modified PBMCs comprise a further
modification to modulate MHC class II expression.
[0498] 147. The plurality of modified PBMCs of embodiment 145,
wherein an innate immune response mounted in an individual in
response to administration, in an allogeneic context, of the
modified PBMCs is reduced compared to an innate immune response
mounted in an individual in response to administration, in an
allogeneic context, of corresponding modified PBMCs that do not
comprise the further modification.
[0499] 148. The plurality of modified PBMCs of any one of
embodiments 1-147, wherein the circulating half-life of the
modified PBMCs in an individual to which they were administered is
increased compared to the circulating half-life of corresponding
modified PBMCs that do not comprise the further modification in an
individual to which they were administered.
[0500] 149. The plurality of modified PBMCs of any one of
embodiments 1-147, wherein the circulating half-life of the
modified PBMCs in an individual to which they were administered is
essentially the same as the circulating half-life of corresponding
modified PBMCs that do not comprise the further modification in an
individual to which they were administered.
[0501] 150. The plurality of modified PBMCs of any one of
embodiments 1-147, wherein the circulating half-life of the
modified PBMCs in an individual to which they were administered is
essentially the same as the circulating half-life of corresponding
unmodified PBMCs.
[0502] 151. The plurality of modified PBMCs of any one of
embodiments 1-150, wherein the plurality of PBMCs comprises one or
more of T cell, B cell, NK cell, monocytes, dendritic cells or NK-T
cells.
[0503] 152. The plurality of modified PBMCs of any one of
embodiments 1-151, wherein the plurality of PBMCs comprises two or
more of T cell, B cell, NK cell, monocytes, dendritic cells or NK-T
cells.
[0504] 153. The plurality of modified PBMCs of any one of
embodiments 1-152, wherein the plurality of PBMCs comprises one or
more of CD3+ T cells, CD20+ B cells, CD14+ monocytes, CD56+NK
cells.
[0505] 154. The plurality of modified PBMCs of any one of
embodiments 10-153, wherein the plurality of input PBMCs comprises
T cells, B cells, NK cells and monocytes, and wherein the ratio of
T cells, B cells, NK cells and monocytes to the total number of
PBMCs in the plurality of input PBMCs is essentially the same as
the ratio of T cells, B cells, NK cells and monocytes to the total
number of PBMCs in whole blood.
[0506] 155. The plurality of modified PBMCs of any one of
embodiments 10-153, wherein the plurality of input PBMCs comprises
T cells, B cells, NK cells and monocytes, and wherein the ratio of
T cells, B cells, NK cells and monocytes to the total number of
PBMCs in the plurality of input PBMCs is essentially the same as
the ratio of T cells, B cells, NK cells and monocytes to the total
number of PBMCs in a leukapheresis product from whole blood.
[0507] 156. The plurality of modified PBMCs of any one of
embodiments 10-153, wherein the plurality of input PBMCs comprises
T cells, B cells, NK cells and monocytes, and wherein the ratio of
T cells, B cells, NK cells and monocytes to the total number of
PBMCs in the plurality of input PBMCs differs by not more than 10%
from the ratio of T cells, B cells, NK cells and monocytes to the
total number of PBMCs in whole blood.
[0508] 157. The plurality of modified PBMCs of any one of
embodiments 10-153, wherein the plurality of input PBMCs comprises
T cells, B cells, NK cells and monocytes, and wherein the ratio of
T cells, B cells, NK cells and monocytes to the total number of
PBMCs in the plurality of input PBMCs differs by not more than 10%
from the ratio of T cells, B cells, NK cells and monocytes to the
total number of PBMCs in a leukapheresis product from whole
blood.
[0509] 158. The plurality of modified PBMCs of any one of
embodiments 10-157, wherein:
(a) at least about 25% of the input PBMCs are T cells; (b) at least
about 2.5% of the input PBMCs are B cells; (c) at least about 3.5%
of the input PBMCs are NK cells; or (d) at least about 4% of the
input PBMCs are monocytes.
[0510] 159. The plurality of modified PBMCs of any one of
embodiments 1-158, wherein:
(a) at least about 20% of the modified PBMCs are T cells; (b) at
least about 2% of the modified PBMCs are B cells; (c) at least
about 3% of the modified PBMCs are NK cells; or (d) at least about
3% of the modified PBMCs are monocytes.
[0511] 160. The plurality of modified PBMCs of any one of
embodiments 1-159, wherein:
(a) not more than about 70% of the input PBMCs are T cells; (b) not
more than about 14% of the input PBMCs are B cells; (c) not more
than about 35% of the input PBMCs are NK cells; or (d) not more
than about 25% of the input PBMCs are monocytes.
[0512] 161. The plurality of modified PBMCs of any one of
embodiments 1-160, wherein:
(a) not more than about 80% of the modified PBMCs are T cells; (b)
not more than about 16% of the modified PBMCs are B cells; (c) not
more than about 40% of the modified PBMCs are NK cells; or (d) not
more than about 30% of the modified PBMCs are monocytes.
[0513] 162. The plurality of modified PBMCs of any one of
embodiments 1-161, wherein:
(a) about 25% to about 70% of the modified PBMCs are T cells; (b)
about 2.5% to about 14% of the modified PBMCs are B cells; (c)
about 3.5% to about 35% of the modified PBMCs are NK cells; or (d)
about 4% to about 25% of the modified PBMCs are monocytes.
[0514] 163. The plurality of modified PBMCs of any one of
embodiments 1-162, wherein:
(a) the percentage of T cells within the plurality of modified
PBMCs and the percentage of T cells within the plurality of input
PBMCs differ by no more than about 10% by number; (b) the
percentage of B cells within the plurality of modified PBMCs and
the percentage of B cells within the plurality of input PBMCs
differ by no more than about 10% by number; (c) the percentage of
NK cells within the plurality of modified PBMCs and the percentage
of NK cells within the plurality of input PBMCs differ by no more
than about 10% by number; and/or (d) the percentage of monocytes
within the plurality of modified PBMCs and the percentage of
monocytes within the plurality of input PBMCs differ by no more
than about 10% by number.
[0515] 164. The plurality of modified PBMCs of any one of
embodiments 3-9 and 12-163, wherein: one or more co-stimulatory
molecules is upregulated in the B cells of the conditioned
plurality of modified PBMCs compared to the B cells in the
plurality of unmodified PBMCs, wherein the co-stimulatory molecule
is CD80 and/or CD86.
[0516] 165. The plurality of modified PBMCs of embodiment 164,
wherein the CD80 and/or CD86 is upregulated in the B cells of the
conditioned plurality of modified PBMCs by more than about
1.2-fold, 1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold,
8-fold, or more than 10-fold compared to the B cells in a plurality
of unconditioned PBMCs.
[0517] 166. The plurality of modified PBMCs of embodiment 164 or
165, wherein the co-stimulatory molecule is CD86.
[0518] 167. The conditioned plurality of modified PBMCs of any one
of embodiments 3-9 and 12-166, wherein the modified PBMCs have
increased expression of one or more of IFN-.gamma., IL-6, MCP-1,
MIP-1.beta., IP-10, or TNF-.alpha. compared to a plurality of
unconditioned PBMCs.
[0519] 168. The conditioned plurality of modified PBMCs of
embodiment 167, wherein the expression of one or more of
IFN-.gamma., IL-6, MCP-1, MIP-1.beta., IP-10, or TNF-.alpha. is
increased by more than about 1.2-fold, 1.5-fold, 1.8-fold, 2-fold,
3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold compared to
the plurality of unconditioned PBMCs.
[0520] 169. A composition comprising the plurality of modified
PBMCs of any one of embodiments 1-168.
[0521] 170. A composition comprising the plurality of modified
PBMCs of any one of embodiments 1-169 for use as a medicament.
[0522] 171. A composition comprising the plurality of modified
PBMCs of any one of embodiments 1-169 for use in a method of
treatment of the human or animal body by surgery, therapy or
diagnosis.
[0523] 172. A composition comprising the plurality of modified
PBMCs of any one of embodiments 1-169 for use in the treatment of a
cancer, an infectious disease or a viral-associated disease.
[0524] 173. The composition of embodiment 172, wherein the cancer
is head and neck cancer, cervical cancer, vulvar cancer, vaginal
cancer, penile cancer, anal cancer, perianal cancer, anogenital
cancer, oral cancer or salivary cancer.
[0525] 174. The composition of any one of embodiments 171-173,
wherein the modified PBMCs is administered prior to, concurrently
with, or following administration of an immune checkpoint
inhibitor.
[0526] 175. The composition of embodiment 174, wherein the immune
checkpoint inhibitor is targeted to any one of PD-1, PD-L1, CTLA-4,
LAG3, TIM-3, TIGIT, VISTA, TIM1, B7-H4 (VTCN1) or BTLA.
[0527] 176. The composition of embodiment 175, wherein the immune
checkpoint inhibitor is targeted to PD-1.
[0528] 177. The composition of embodiment 175, wherein the immune
checkpoint inhibitor is targeted to PD-L1.
[0529] 178. The composition of any one of embodiments 171-177,
wherein the modified PBMCs is administered prior to, concurrently
with, or following administration of a therapeutic agent.
[0530] 179. The composition of embodiment 178, wherein the
therapeutic agent is a chemotherapeutic agent.
[0531] 180. The composition of embodiment 172, wherein the
infectious disease is associated with HIV, HPV, EBV, MCV, HBV or
HCV.
[0532] 181. A pharmaceutical composition comprising the modified
PBMCs of any one of embodiments 1-168 and a pharmaceutically
acceptable carrier.
[0533] 182. The composition of embodiment any one of embodiments
171-181, wherein the composition is for treatment of cancers or
infectious diseases.
[0534] 183. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use as a medicament,
wherein the conditioned plurality of modified PBMCs is prepared by
a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0535] 184. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use as a medicament,
wherein the conditioned plurality of modified PBMCs is prepared by
a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; and c) incubating the plurality of
modified PBMCs comprising the nucleic acid encoding the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the nucleic acid encoding the antigen to condition,
wherein the nucleic acid is expressed in the PBMCs to produce the
antigen, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0536] 185. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treatment of the human or animal body by surgery, therapy or
diagnosis, wherein the conditioned plurality of modified PBMCs is
prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0537] 186. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treatment of the human or animal body by surgery, therapy or
diagnosis, wherein the conditioned plurality of modified PBMCs is
prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding antigen; and c) incubating the plurality of modified
PBMCs comprising the nucleic acid encoding the antigen with an
adjuvant for a sufficient time for the modified PBMCs comprising
the nucleic acid encoding the antigen to condition, wherein the
nucleic acid is expressed in the PBMCs to produce the antigen,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen.
[0538] 187. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use as a medicament,
wherein the conditioned plurality of modified PBMCs is prepared by
a process comprising the steps of:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; and c) incubating the
conditioned plurality of perturbed input PBMCs with the antigen for
a sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0539] 188. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use as a medicament,
wherein the conditioned plurality of modified PBMCs is prepared by
a process comprising the steps of:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a conditioned plurality of perturbed input PBMCs; and c)
incubating the conditioned plurality of perturbed input PBMCs with
the nucleic acid encoding the antigen for a sufficient time to
allow the nucleic acid encoding the antigen to enter the perturbed
input PBMCs, wherein the nucleic acid is expressed in the PBMCs to
produce the antigen, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen.
[0540] 189. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treatment of the human or animal body, wherein the conditioned
plurality of modified PBMCs is prepared by a process comprising the
steps of:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; and c) incubating the
conditioned plurality of perturbed input PBMCs with the antigen for
a sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0541] 190. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treatment of the human or animal body, wherein the conditioned
plurality of modified PBMCs is prepared by a process comprising the
steps of:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a conditioned plurality of perturbed input PBMCs; and c)
incubating the conditioned plurality of perturbed input PBMCs with
the nucleic acid encoding the antigen for a sufficient time to
allow the nucleic acid encoding the antigen to enter the perturbed
input PBMCs, wherein the nucleic acid is expressed in the PBMCs to
produce the antigen, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen.
[0542] 191. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treating cancer an infectious disease or a viral associated disease
in an individual, wherein the conditioned plurality of modified
PBMCs is prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0543] 192. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treating cancer an infectious disease or a viral associated disease
in an individual, wherein the conditioned plurality of modified
PBMCs is prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; and c) incubating the plurality of
modified PBMCs comprising the nucleic acid encoding the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the nucleic acid encoding the antigen to condition,
wherein the nucleic acid is expressed in the PBMCs to produce the
antigen, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0544] 193. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in the treatment of
cancer, an infectious disease or a viral associated disease in an
individual, wherein the conditioned plurality of modified PBMCs is
prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0545] 194. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in the treatment of
cancer, an infectious disease or a viral associated disease in an
individual, wherein the conditioned plurality of modified PBMCs is
prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; and c) incubating the plurality of
modified PBMCs comprising the nucleic acid encoding antigen with an
adjuvant for a sufficient time for the modified PBMCs comprising
the nucleic acid encoding the antigen to condition, wherein the
nucleic acid is expressed in the PBMCs to produce the antigen,
thereby generating the conditioned plurality of modified PBMCs
comprising the antigen.
[0546] 195. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treating a HPV-associated disease in an individual, wherein the
conditioned plurality of modified PBMCs is prepared by a process
comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0547] 196. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in a method of
treating a HPV-associated disease in an individual, wherein the
conditioned plurality of modified PBMCs is prepared by a process
comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; and c) incubating the plurality of
modified PBMCs comprising the nucleic acid encoding the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the nucleic acid encoding the antigen to condition,
wherein the nucleic acid is expressed in the PBMCs to produce the
antigen, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0548] 197. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in the treatment of a
HPV-associated disease in an individual, wherein the conditioned
plurality of modified PBMCs is prepared by a process comprising the
steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0549] 198. A composition comprising a conditioned plurality of
modified PBMCs comprising an antigen for use in the treatment of a
HPV-associated disease in an individual, wherein the conditioned
plurality of modified PBMCs is prepared by a process comprising the
steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; and c) incubating the plurality of
modified PBMCs comprising the antigen with an adjuvant for a
sufficient time for the modified PBMCs comprising the nucleic acid
encoding the antigen to condition, wherein the nucleic acid is
expressed in the PBMCs to produce the antigen, thereby generating
the conditioned plurality of modified PBMCs comprising the
antigen.
[0550] 199. Use of a composition comprising a conditioned plurality
of modified PBMCs comprising an antigen in the manufacture of a
medicament for treating cancer, an infectious disease or a
viral-associated disease in an individual, wherein the conditioned
plurality of modified PBMCs is prepared by a process comprising the
steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0551] 200. Use of a composition comprising a conditioned plurality
of modified PBMCs comprising an antigen in the manufacture of a
medicament for treating a HPV-associated disease, wherein the
conditioned plurality of modified PBMCs is prepared by a process
comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0552] 201. A conditioned plurality of modified PBMCs comprising a
human papillomavirus (HPV) antigen, prepared by a process
comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 4 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, thereby generating the conditioned plurality of
modified PBMCs comprising the HPV antigen.
[0553] 202. A conditioned plurality of modified PBMCs comprising a
HPV antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 4 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, wherein the CpG ODN is CpG 7909, thereby generating
the conditioned plurality of modified PBMCs comprising the HPV
antigen.
[0554] 203. A conditioned plurality of modified PBMCs comprising a
HPV antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 4 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for
about 1 hour to about 24 hours for the modified PBMCs comprising
the HPV antigen to condition, thereby generating the conditioned
plurality of modified PBMCs comprising the HPV antigen.
[0555] 204. A conditioned plurality of modified PBMCs comprising a
HPV antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 4 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for
about 1 hour to about 24 hours for the modified PBMCs comprising
the HPV antigen to condition, wherein the CpG ODN is CpG 7909,
thereby generating the conditioned plurality of modified PBMCs
comprising the HPV antigen.
[0556] 205. The conditioned plurality of the modified PBMCs of any
one of embodiments 201-204, wherein the diameter of the
constriction is (a) about 4.2 .mu.m to about 6 .mu.m; or (b) about
4.5 .mu.m.
[0557] 206. The conditioned plurality of the modified PBMCs of any
one of embodiments 201-205, wherein the plurality of modified PBMCs
comprising the HPV antigen is incubated with a CpG ODN for (a)
about 2 hour to about 10 hours; (b) about 3 hours to about 6 hours;
or (c) about 4 hours.
[0558] 207. A conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for a sufficient time
for the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen.
[0559] 208. A conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for a sufficient time
for the modified PBMCs comprising the antigen to condition, wherein
the CpG ODN is CpG 7909, thereby generating the conditioned
plurality of modified PBMCs comprising the antigen.
[0560] 209. A conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for about 1 hour to
about 24 hours for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0561] 210. A conditioned plurality of modified PBMCs comprising an
antigen, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for about 1 hour to
about 24 hours for the modified PBMCs comprising the antigen to
condition, wherein the CpG ODN is CpG 7909, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0562] 211. The conditioned plurality of the modified PBMCs of any
one of embodiments 207-210, wherein the diameter of the
constriction is about 4 .mu.m to about 10 .mu.m.
[0563] 212. The conditioned plurality of the modified PBMCs of any
one of embodiments 207-211, the diameter of the constriction is
about 3 .mu.m to about 6 .mu.m.
[0564] 213. The conditioned plurality of the modified PBMCs of any
one of embodiments 207-212, wherein the diameter of the
constriction is (a) about 4.2 .mu.m to about 6 .mu.m; or (b) about
4.5 .mu.m.
[0565] 214. The conditioned plurality of the modified PBMCs of any
one of embodiments 207-213, wherein the plurality of modified PBMCs
comprising the antigen is incubated with a CpG ODN for (a) about 2
hour to about 10 hours; (b) about 3 hours to about 6 hours; or (c)
about 4 hours.
[0566] 215. A method for stimulating an immune response in an
individual, comprising administering to the individual the
plurality of modified PBMCs of any one of embodiments 1-168, the
composition of embodiment 169-180, or the pharmaceutical
composition of embodiment 181.
[0567] 216. A method for stimulating an immune response in an
individual, comprising:
a) administering a plurality of modified PBMCs comprising an
antigen comprising the amino acid sequence of any one of SEQ ID
NOs: 18-25 to the individual; and b) administering an adjuvant to
the individual.
[0568] 217. A method for stimulating an immune response in an
individual, comprising:
a) incubating a plurality of PBMCs comprising an antigen with an
adjuvant for a sufficient time for the PBMCs to condition, thereby
generating a conditioned plurality of PBMCs comprising the antigen;
b) administering the conditioned plurality of PBMCs comprising the
antigen to the individual.
[0569] 218. A method for stimulating an immune response in an
individual, comprising:
a) incubating a plurality of PBMCs with an adjuvant for a
sufficient time for the PBMCs to condition, thereby generating a
conditioned plurality of PBMCs comprising the antigen; b)
introducing an antigen to the plurality of PBMCs; and c)
administering the conditioned plurality of PBMCs comprising the
antigen to the individual.
[0570] 219. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; c) incubating
the plurality of modified PBMCs comprising the antigen with an
adjuvant for a sufficient time for the modified PBMCs comprising
the antigen to condition, thereby generating a conditioned
plurality of modified PBMCs comprising the antigen; and d)
administering the conditioned plurality of modified PBMCs
comprising the antigen to the individual.
[0571] 220. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding an antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; c) incubating the plurality of modified
PBMCs comprising the nucleic acid encoding the antigen with an
adjuvant for a sufficient time for the modified PBMCs comprising
the nucleic acid encoding the antigen to condition, wherein the
nucleic acid is expressed in the PBMCs to produce the antigen,
thereby generating a conditioned plurality of modified PBMCs
comprising the antigen; and d) administering the conditioned
plurality of modified PBMCs comprising the antigen to the
individual.
[0572] 221. The method of embodiment 220, further comprising
isolating the plurality of modified PBMCs comprising the antigen
from the cell suspension before incubation with the adjuvant.
[0573] 222. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an antigen and an adjuvant to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the antigen and the adjuvant for a
sufficient time to allow the antigen and the adjuvant to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the antigen and adjuvant; and c) administering the
plurality of modified PBMCs to the individual.
[0574] 223. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nuclein acid encoding an antigen and for an adjuvant
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the nucleic
acid encoding the antigen and with the adjuvant for a sufficient
time to allow the nucleic acid encoding the antigen and the
adjuvant to enter the perturbed input PBMCs, wherein the nucleic
acid is expressed in the PBMCs to produce the antigen, thereby
generating a plurality of modified PBMCs comprising the antigen and
adjuvant; and c) administering the plurality of modified PBMCs
comprising the antigen and the adjuvant to the individual.
[0575] 224. The method of embodiment 223, wherein the concentration
of the antigen incubated with the perturbed input PBMCs is between
about 0.1 .mu.M and about 1 mM and/or the concentration of the
adjuvant incubated with the perturbed input PBMCs is between about
0.1 .mu.M and about 1 mM.
[0576] 225. The method of any one of embodiments 222-224, wherein
the concentration of the antigen incubated with the perturbed input
PBMCs is between about 0.1 .mu.M and about 10 .mu.M and/or the
concentration of the adjuvant incubated with the perturbed input
PBMCs is between about 0.1 .mu.M and about 10 .mu.M.
[0577] 226. The method of any one of embodiments 222-225, wherein
the concentration of the antigen incubated with the perturbed input
PBMCs is about 1 .mu.M and/or the concentration of the adjuvant
incubated with the perturbed input PBMCs is about 1 .mu.M.
[0578] 227. The method of any one of embodiments 222-226, wherein
the ratio of the antigen to the adjuvant incubated with the
perturbed input PBMCs is between about 10000:1 to about
1:10000.
[0579] 228. The method of any one of embodiments 222-227, wherein
the ratio of the antigen to the adjuvant incubated with the
perturbed input PBMCs is about 200:1.
[0580] 229. A method for stimulating an immune response in an
individual, comprising:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; c) incubating the conditioned
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen; and d) administering the conditioned
plurality of modified PBMCs to the individual.
[0581] 230. The method of embodiment 229, wherein the concentration
of the adjuvant incubated with the input PBMCs is between about 0.1
.mu.M and about 1 mM.
[0582] 231. The method of embodiment 229 or 230, wherein the
concentration of the adjuvant incubated with the input PBMCs is
between about 0.1 .mu.M and about 10 .mu.M.
[0583] 232. The method of any one of embodiments 229-231, wherein
the concentration of the adjuvant incubated with the input PBMCs is
about 1 .mu.M.
[0584] 233. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
comprising an adjuvant through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating a plurality of modified PBMCs comprising
the antigen and the adjuvant; and c) administering the plurality of
modified PBMCs to the individual.
[0585] 234. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising an input PBMCs comprising
an antigen through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for an adjuvant to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the adjuvant for a sufficient time to
allow the adjuvant to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen and
the adjuvant; and c) administering the plurality of modified PBMCs
to the individual.
[0586] 235. The method of embodiment 234, wherein the concentration
of the adjuvant incubated with the perturbed input PBMCs is between
about 0.1 .mu.M and about 1 mM.
[0587] 236. The method of embodiment 234 or 235, wherein the
concentration of the adjuvant incubated with the perturbed input
PBMCs is between about 0.1 .mu.M and about 10 .mu.M.
[0588] 237. The method of any one of embodiments 234-236, wherein
the concentration of the adjuvant incubated with the perturbed
input PBMCs is about 1 .mu.M.
[0589] 238. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for an antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; c)
administering the plurality of modified PBMCs to the individual;
and d) administering an adjuvant to the individual.
[0590] 239. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising an input PBMCs comprising
an antigen through a cell-deforming constriction, wherein a
diameter of the constriction is a function of a diameter of the
input PBMCs in the suspension, thereby causing perturbations of the
input PBMCs large enough for an adjuvant to pass through to form a
plurality of perturbed input PBMCs; b) incubating the plurality of
perturbed input PBMCs with the adjuvant for a sufficient time to
allow the adjuvant to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the antigen and
the adjuvant; and c) administering the plurality of modified PBMCs
to the individual; and d) administering an adjuvant to the
individual.
[0591] 240. The method of any one of embodiments 219-233, 238 and
239, wherein the concentration of the antigen incubated with the
perturbed input PBMCs is between about 0.1 .mu.M and about 1
mM.
[0592] 241. The method of any one of embodiments 219-233 and
238-240, wherein the concentration of the antigen incubated with
the perturbed input PBMCs is between about 0.1 .mu.M and about 10
.mu.M.
[0593] 242. The method of any one of embodiments 219-233 and
238-241, wherein the concentration of the antigen incubated with
the perturbed input PBMCs is about 1 .mu.M.
[0594] 243. The method of any one of embodiments 222-228 and
233-242, wherein the process further comprises:
incubating the plurality of modified PBMCs comprising the antigen
and/or adjuvant with a second adjuvant for a sufficient time for
the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen and/or adjuvant.
[0595] 244 The method of embodiment 243, wherein the concentration
of the second adjuvant incubated with the modified PBMCs is between
about 0.1 .mu.M and about 1 mM.
[0596] 245. The method of embodiment 243 or 244, wherein the
concentration of the second adjuvant incubated with the modified
PBMCs is between about 0.1 .mu.M and about 10 .mu.M.
[0597] 246. The plurality of modified PBMCs of any one of
embodiments 243-245, wherein the concentration of the second
adjuvant incubated with the modified PBMCs is about 1 .mu.M.
[0598] 247. A method for stimulating an immune response in an
individual, comprising:
administering to the individual a plurality of PBMCs associated
with an antigen, wherein the plurality of modified PBMCs is
prepared by a process comprising the steps of: a) incubating a
plurality of input PBMCs with an antigen for a sufficient time to
allow the antigen to associate with the cell surface of the input
PBMCs, thereby generating the plurality of PBMCs associated with
the antigen; and b) administering the plurality of modified PBMCs
to the individual.
[0599] 248. The method of any one of embodiments 215-247, further
comprising administering an adjuvant to the individual.
[0600] 249. The method of embodiment 248, wherein the adjuvant is
administered before, concurrently with, or after administration of
the plurality of modified PBMCs to the individual.
[0601] 250. A plurality of PBMCs comprising an antigen for use in a
method of stimulating an immune response in an individual according
to any one of embodiments 215-245 and 247-249.
[0602] 251. A method for generating a conditioned plurality of
PBMCs comprising an antigen, comprising incubating a plurality of
PBMCs comprising the antigen with an adjuvant for a sufficient time
for the PBMCs to condition, thereby generating the conditioned
plurality of PBMCs comprising the antigen.
[0603] 252. A method for generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0604] 253. A method for generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, thereby
generating a plurality of modified PBMCs comprising the nucleic
acid encoding the antigen; and c) incubating the plurality of
modified PBMCs comprising the nucleic acid encoding the antigen
with an adjuvant for a sufficient time for the modified PBMCs
comprising the nucleic acid encoding the antigen to condition,
wherein the nucleic acid is expressed in the PBMCs to produce the
antigen, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0605] 254. The method of embodiment 252, further comprising
isolating the plurality of modified PBMCs comprising the antigen
from the cell suspension before incubation with the adjuvant.
[0606] 255. The method of embodiment 253, further comprising
isolating the plurality of modified PBMCs comprising the nucleic
acid encoding the antigen from the cell suspension before
incubation with the adjuvant.
[0607] 256. A method for generating a plurality of modified PBMCs
comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; and b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating the
plurality of modified PBMCs comprising the antigen.
[0608] 257. A method for generating a plurality of modified PBMCs
comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a plurality of perturbed input PBMCs; and b) incubating the
plurality of perturbed input PBMCs with the nucleic acid encoding
the antigen for a sufficient time to allow the nucleic acid
encoding the antigen to enter the perturbed input PBMCs, wherein
the nucleic acid is expressed in the PBMCs to produce the antigen,
thereby generating the plurality of modified PBMCs comprising the
antigen.
[0609] 258. A method for generating a plurality of modified PBMCs
comprising an antigen and an adjuvant, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen and the adjuvant to pass through to form a
plurality of perturbed input PBMCs; and b) incubating the plurality
of perturbed input PBMCs with the antigen and the adjuvant for a
sufficient time to allow the antigen and the adjuvant to enter the
perturbed input PBMCs, thereby generating the plurality of modified
PBMCs comprising the antigen and adjuvant.
[0610] 259. A method for generating a plurality of modified PBMCs
comprising an antigen and an adjuvant, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen and for the adjuvant
to pass through to form a plurality of perturbed input PBMCs; and
b) incubating the plurality of perturbed input PBMCs with the
nucleic acid encoding the antigen and with the adjuvant for a
sufficient time to allow the nucleic acid encoding the antigen and
the adjuvant to enter the perturbed input PBMCs, wherein the
nucleic acid is expressed in the PBMCs to produce the antigen,
thereby generating the plurality of modified PBMCs comprising the
antigen and adjuvant.
[0611] 260. A method of generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a conditioned
plurality of perturbed input PBMCs; and c) incubating the
conditioned plurality of perturbed input PBMCs with the antigen for
a sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0612] 261. A method of generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) incubating a plurality of input PBMCs with an adjuvant for a
sufficient time for the input PBMCs to condition, thereby
generating a conditioned plurality of input PBMCs; b) passing a
cell suspension comprising the conditioned plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for a nucleic acid encoding the antigen to pass through to
form a conditioned plurality of perturbed input PBMCs; and c)
incubating the conditioned plurality of perturbed input PBMCs with
the nucleic acid encoding the antigen for a sufficient time to
allow the nucleic acid encoding the antigen to enter the perturbed
input PBMCs, wherein the nucleic acid is expressed in the PBMCs to
produce the antigen, thereby generating the conditioned plurality
of modified PBMCs comprising the antigen.
[0613] 262. A method for generating a plurality of modified PBMCs
comprising an antigen and an adjuvant, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
comprising an adjuvant through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an antigen to pass through to
form a plurality of perturbed input PBMCs; and b) incubating the
plurality of perturbed input PBMCs with the antigen for a
sufficient time to allow the antigen to enter the perturbed input
PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen and the adjuvant.
[0614] 263. A method for generating a plurality of modified PBMCs
comprising an antigen and an adjuvant, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
comprising an adjuvant through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for a nucleic acid encoding an
antigen to pass through to form a plurality of perturbed input
PBMCs; and b) incubating the plurality of perturbed input PBMCs
with the nucleic acid encoding the antigen for a sufficient time to
allow the nucleic acid encoding the antigen to enter the perturbed
input PBMCs, wherein the nucleic acid is expressed in the PBMCs to
produce the antigen, thereby generating the plurality of modified
PBMCs comprising the antigen and the adjuvant.
[0615] 264. A method for generating a plurality of modified PBMCs
comprising an antigen and an adjuvant, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
comprising an antigen through a cell-deforming constriction,
wherein a diameter of the constriction is a function of a diameter
of the input PBMCs in the suspension, thereby causing perturbations
of the input PBMCs large enough for an adjuvant to pass through to
form a plurality of perturbed input PBMCs; and b) incubating the
plurality of perturbed input PBMCs with the adjuvant for a
sufficient time to allow the adjuvant to enter the perturbed input
PBMCs, thereby generating the plurality of modified PBMCs
comprising the antigen and the adjuvant.
[0616] 265. The method of any one of embodiments 219-245, 247-248
and 251-264 wherein the process further comprises a step of
incubating the input PBMCs and/or the modified PBMCs with an agent
that enhances the viability and/or function of the modified PBMCs
as compared to corresponding modified PBMCs prepared without the
further incubation step.
[0617] 266. The method of any one of embodiments 219-245, 247-249
and 251-265, wherein the diameter of the constriction is about 10%
to about 99% of the mean diameter of the plurality of input
PBMCs.
[0618] 267. The method of any one of embodiments 219-245, 247-249
and 251-265, wherein the diameter of the constriction is about 10%
to about 70% of the mean diameter of the plurality of input
PBMCs.
[0619] 268. The method of any one of embodiments 219-245, 247-249
and 251-267, wherein the diameter of the constriction is about 20%
to about 60% of the mean diameter of the plurality of input
PBMCs.
[0620] 269. The method of any one of embodiments 219-245, 247-249
and 251-268, wherein the diameter of the constriction is about 30%
to about 45% of the mean diameter of the plurality of input
PBMCs.
[0621] 270. The method of any one of embodiments 219-245, 247-249
and 251-269, wherein the diameter of the constriction is about 10%
to about 99% of the mean diameter of the subpopulation of cells
having the smallest diameter within the plurality of input
PBMCs.
[0622] 271. The method of any one of embodiments 219-245, 247-249
and 251-270, wherein the diameter of the constriction is about 10%
to about 70% of the mean diameter of a subpopulation of cells
having the smallest diameter within the plurality of input
PBMCs.
[0623] 272. The method of any one of embodiments 219-245, 247-249
and 251-271, wherein the diameter of the constriction is about 20%
to about 60% of the mean diameter of the subpopulation of cells
having the smallest diameter within the plurality of input
PBMCs.
[0624] 273. The method of any one of embodiments 219-245, 247-249
and 251-272, wherein the diameter of the constriction is about 30%
to about 45% of the mean diameter of the subpopulation of cells
having the smallest diameter within the plurality of input
PBMCs.
[0625] 274. The method of any one of embodiments 219-245, 247-249
and 251-270, wherein the diameter of the constriction is about 50%
to about 99% of the mean diameter of a subpopulation of cells
having the smallest diameter within the plurality of input
PBMCs.
[0626] 275. The method of any one of embodiments 219-245, 247-249,
251-270 and 274, wherein the diameter of the constriction is about
50% to about 90% of the mean diameter of a subpopulation of cells
having the smallest diameter within the plurality of input
PBMCs.
[0627] 276. The method of any one of embodiments 219-245, 247-249,
251-270 and 274-275, wherein the diameter of the constriction is
about 50% to about 80% of the mean diameter of a subpopulation of
cells having the smallest diameter within the plurality of input
PBMCs.
[0628] 277. The method of any one of embodiments 219-245, 247-249,
251-270 and 274-276, wherein the diameter of the constriction is
about 50% to about 70% of the mean diameter of a subpopulation of
cells having the smallest diameter within the plurality of input
PBMCs.
[0629] 278. The method of any one of embodiments 219-245, 247-249,
and 251-270, wherein the diameter of the constriction is about 60%
to about 90% of the mean diameter of a subpopulation of cells
having the smallest diameter within the plurality of input PBMCs.
1
[0630] 279. The method of any one of embodiments 219-245, 247-249,
251-270 and 278, wherein the diameter of the constriction is about
60% to about 80% of the mean diameter of a subpopulation of cells
having the smallest diameter within the plurality of input
PBMCs.
[0631] 280. The method of any one of embodiments 219-245, 247-249,
251-270 and 278-279, wherein the diameter of the constriction is
about 60% to about 70% of the mean diameter of a subpopulation of
cells having the smallest diameter within the plurality of input
PBMCs.
[0632] 281. The method of any one of embodiments 219-245, 247-249,
and 251-280, wherein the diameter of the constriction is about 10%
to about 99% of the mean diameter of the subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0633] 282. The method of any one of embodiments 219-245, 247-249,
and 251-281 wherein the diameter of the constriction is about 10%
to about 70% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0634] 283. The method of any one of embodiments 219-245, 247-249,
and 251-282, wherein the diameter of the constriction is about 20%
to about 60% of the mean diameter of the subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0635] 284. The method of any one of embodiments 219-245, 247-249,
and 251-283, wherein the diameter of the constriction is about 20%
to about 30% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0636] 285. The method of any one of embodiments 219-245, 247-249,
and 251-284, wherein the diameter of the constriction is about 20%
to about 25% of the mean diameter of a subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0637] 286. The method of any one of embodiments 219-245, 247-249,
and 251-283, wherein the diameter of the constriction is about 30%
to about 45% of the mean diameter of the subpopulation of cells
having the largest diameter within the plurality of input
PBMCs.
[0638] 287. The method of any one of embodiments 270-286, wherein
the subpopulation of cells having the smallest diameter within the
plurality of input PBMCs are T cells.
[0639] 288. The method of any one of embodiments 281-286, wherein
the subpopulation of cells having the largest diameter within the
plurality of input PBMCs are monocytes.
[0640] 289. The method of any one of embodiments 219-245, 247-249
and 251-275, wherein the diameter of the constriction is about 3
.mu.m to about 10 .mu.m.
[0641] 290. The method of any one of embodiments 219-245, 247-249
and 251-289, wherein the diameter of the constriction is about 3
.mu.m to about 6 .mu.m.
[0642] 291. The method of any one of embodiments 219-245, 247-249
and 251-289, wherein the diameter of the constriction is about 4
.mu.m to about 10 .mu.m.
[0643] 292. The method of any one of embodiments 219-245, 247-249
and 251-291, wherein the diameter of the constriction is about 4.2
.mu.m to about 6 .mu.m.
[0644] 293. The method of any one of embodiments 219-245, 247-249
and 251-292, wherein the diameter of the constriction is about 4.5
.mu.m.
[0645] 294. The method of any one of embodiments 219-245, 247-249
and 251-293, wherein the plurality of input PBMCs is passed through
the constriction under a pressure ranging from about 30 psi to
about 90 psi.
[0646] 295. The method of any one of embodiments 219-245, 247-249
and 251-293, wherein the plurality of input PBMCs is passed through
the constriction under a pressure ranging from about 207 kPa to
about 830 kPa or about 415 kPa to about 621 kPa.
[0647] 296. The method of any one of embodiments 219-245, 247-249
and 251-295, wherein the plurality of input PBMCs is passed through
the constriction at a flow rate between about 0.001 mL/cm.sup.2/sec
to about 200 L/cm.sup.2/sec.
[0648] 297. The method of any one of embodiments 219-245, 247-249
and 251-295, wherein the plurality of input PBMCs is passed through
the constriction at a flow rate between about 0.1 mL/cm.sup.2/sec
to about 150 L/cm.sup.2/sec.
[0649] 298. The method of any one of embodiments 219-245, 247-249
and 251-297, wherein the plurality of input PBMCs is passed through
the constriction at a flow rate or about 100 L/cm.sup.2/sec.
[0650] 299. The method of any one of embodiments 219-245, 247-249
and 251-298, wherein the plurality of input PBMCs is passed through
the constriction at a temperature ranging from about 0.degree. C.
to about 37.degree. C.
[0651] 300. The method of any one of embodiments 219-245, 247-249
and 251-299, wherein subsequent to passing through the constriction
the plurality of modified PBMCs is incubated at a temperature of
about 37.degree. C. for a sufficient time to allow the modified
PBMCs to normalize to about 37.degree. C.
[0652] 301. The method of any one of embodiments 219-245, 247-249
and 251-300, wherein subsequent to passing through the constriction
the plurality of modified PBMCs is incubated at a temperature of
about 25.degree. C. for a sufficient time to allow the modified
PBMCs to normalize to about 25.degree. C.
[0653] 302. The method of any one of embodiments 219-245, 247-249
and 251-301, wherein the cross-sectional shape of the constriction
is selected from the group consisting of: circular, elliptical,
round, square, rectangular, star-shaped, triangular, polygonal,
pentagonal, hexagonal, heptagonal, and octagonal.
[0654] 303. The method of any one of embodiments 219-245, 247-249
and 251-302, wherein the cross-sectional shape of the constriction
is a slit.
[0655] 304. The method of embodiment 303, wherein slit comprises a
width of about 3 .mu.m-6 .mu.m and/or a depth of about 20 .mu.m-120
.mu.m.
[0656] 305. The method of embodiment 303, wherein the slit
comprises a width of about 4.2 .mu.m-6 .mu.m and/or a depth of
about 20-120 .mu.m.
[0657] 306. The method of embodiment 303-305, wherein the slit
comprises a width of 4.5 .mu.m and/or a depth of 80 .mu.m.
[0658] 307. The method of any one of embodiments 219-245, 247-249
and 251-306, wherein the cell suspension comprising the plurality
of input PBMCs is passed through multiple constrictions wherein the
multiple constrictions are arranged in series and/or in
parallel.
[0659] 308. The method of any one of embodiments 219-245, 247-249
and 251-307, wherein the constriction comprises an entrance portion
and an exit portion, wherein:
(a) the entrance portion defines an entrance angle and the entrance
angle is between about 0 degree to about 90 degrees or about 20-22
degrees; and/or (b) the exit portion defines an exit angle and the
exit angle is between about 0 degree to about 90 degrees or about
20-22 degrees; preferably about 20-22 degrees for (a) and (b).
[0660] 309. The method of any one of embodiments 219-245, 247-249
and 251-308, wherein the cell suspension comprising the plurality
of input PBMCs are passed through multiple constrictions, wherein
the multiple constrictions are arranged in series and/or in
parallel.
[0661] 310. The method of any one of embodiments 219-221, 229-232,
243-245, 247-249, 251 and 265-309, wherein the plurality of
modified PBMCs is incubated with the adjuvant for about 1 to about
24 hours for the modified PBMCs to condition.
[0662] 311. The method of any one of embodiments 219-221, 229-232,
243-245, 251 and 265-310, wherein the plurality of modified PBMCs
is incubated with the adjuvant for about 2 to about 10 hours for
the modified PBMCs to condition.
[0663] 312. The method of any one of embodiments 219-221, 229-232,
243-245, 251 and 265-311, wherein the plurality of modified PBMCs
is incubated with the adjuvant for about 3 to about 6 hours for the
modified PBMCs to condition.
[0664] 313. The method of any one of embodiments 219-221, 229-232,
243-245, 251 and 265-312, wherein the plurality of modified PBMCs
is incubated with the adjuvant for about 4 hours for the modified
PBMCs to condition.
[0665] 314. The method of any one of embodiments 215-245, 247-249
and 251-313, wherein the antigen and/or adjuvant are present in the
cytosol and/or a vesicle of a cell in plurality of modified
PBMCs.
[0666] 315. The method of embodiment 314, wherein the vesicle is an
endosome.
[0667] 316. The method of any one of embodiments 215-245, 247-249
and 251-315, wherein the antigen and/or the adjuvant are present in
multiple compartments of a cell in plurality of modified PBMCs.
[0668] 317. The method of any one of embodiments 222-228, 233-237,
247-249, and 251-316, wherein the antigen is present in the cytosol
and the adjuvant is present in a vesicle of a cell in the plurality
of modified PBMCs.
[0669] 318. The method of any one of embodiments 215-245, 247-249
and 251-317, wherein the antigen is bound to the surface of a cell
in plurality of modified PBMCs.
[0670] 319. The method of any one of embodiments 215-245, 247-249
and 251-318, wherein the adjuvant is a CpG oligodeoxynucleotide
(ODN), LPS, IFN-.alpha., STING agonists, RIG-I agonists, poly I:C,
R837, R848, a TLR3 agonist, a TLR4 agonist or a TLR 9 agonist
[0671] 320. The method of embodiment 319, wherein the adjuvant is a
CpG ODN.
[0672] 321. The method of embodiment 320, wherein the CpG ODN is a
Class A CpG ODN, a Class B CpG ODN, or a Class C CpG ODN.
[0673] 322. The method of any one of embodiments 215-245, 247-249
and 251-315, wherein the antigen is a disease-associated
antigen.
[0674] 323. The method of embodiment 322, wherein the antigen is
derived from peptides or mRNA isolated from a diseased cell.
[0675] 324. The method of any one of embodiments 215-245, 247-249
and 251-323, wherein the antigen is a non-self antigen.
[0676] 325. The method of any one of embodiments 215-245, 247-249
and 251-324, wherein the antigen is a tumor antigen, viral antigen,
bacterial antigen, or fungal antigen.
[0677] 326. The method of any one of embodiment 215-245, 247-249
and 251-325, wherein the antigen is derived from a tumor
lysate.
[0678] 327. The method of embodiment 325, wherein the antigen is a
human papillomavirus (HPV) antigen.
[0679] 328. The method of embodiment 327, wherein the HPV is HPV-16
or HPV-18.
[0680] 329. The method of embodiment 327 or 328, wherein the
antigen comprises an HLA-A2-restricted peptide derived from HPV E6
and/or E7.
[0681] 330. The method of embodiment 329, wherein the
HLA-A2-restricted peptide comprises the amino acid sequence of any
one of SEQ ID NOs: 1-4.
[0682] 331. The method of embodiment 330, wherein the antigen
comprises the amino acid sequence of any one of SEQ ID NOs:
18-25.
[0683] 332. The method of any one of embodiments 215-245, 247-249
and 251-315, wherein the modified PBMCs comprise a plurality of
antigens that comprise a plurality of immunogenic epitopes.
[0684] 333. The method of embodiment 332, wherein following
administration to an individual of the modified PBMCs comprising
the plurality of antigens that comprise the plurality of
immunogenic epitopes, none of the plurality of immunogenic epitopes
decreases an immune response in the individual to any of the other
immunogenic epitopes.
[0685] 334. The method of any one of embodiments 215-245, 247-249
and 251-333, wherein the antigen is a polypeptide comprising an
immunogenic peptide epitope.
[0686] 335. The method of embodiment 334, wherein the immunogenic
peptide epitope is fused to an N-terminal flanking polypeptide
and/or a C-terminal flanking polypeptide.
[0687] 336. The method of embodiment 334 or 335, wherein the
antigen is a polypeptide comprising an immunogenic peptide epitope
and one or more heterologous peptide sequences.
[0688] 337. The method of embodiment 334, wherein the antigen is a
polypeptide comprising an immunogenic peptide epitope that is
flanked on the N-terminus and/or the C-terminus by heterologous
peptide sequences.
[0689] 338. The method of any one of embodiments 334-337, wherein
the flanking heterologous peptide sequences are derived from a
disease-associated immunogenic peptides.
[0690] 339. The method of any one of embodiments 334-337, wherein
the N-terminal flanking polypeptide comprises the amino acid
sequence of any one of SEQ ID NOs: 5-10 and/or the C-terminal
flanking polypeptide comprises the amino acid sequence of any one
of SEQ ID NOs: 11-17.
[0691] 340. The method of any one of embodiments 215-245, 247-249
and 251-323, wherein the antigen is capable of being processed into
an MHC class I-restricted peptide and/or an MHC class II-restricted
peptide.
[0692] 341. The method of any one of embodiments 217, 222-227, 233,
237, 239-245, 247-249 and 251-340, wherein the modified PBMCs
comprise the adjuvant at a concentration between about 0.1 .mu.M
and about 1 mM.
[0693] 342. The method of any one of embodiments 215-245, 247-249
and 251-325, wherein the modified PBMCs comprise the antigen at a
concentration between about 0.1 .mu.M and about 1 mM.
[0694] 343. The method of any one of embodiments 217, 222-227, 233,
237, 239-245, 247-249 and 251-342, wherein the ratio of the antigen
to the adjuvant is between about 10000:1 to about 1:10000.
[0695] 344. The method of embodiment 317, wherein the ratio of the
antigen to the adjuvant is about 200:1.
[0696] 345. The method of any one of embodiments 215-245, 247-249
and 251-344, wherein the modified PBMCs comprise a complex
comprising: a) the antigen, b) the antigen and at least one other
antigen, c) the antigen and the adjuvant, d) the nucleic acid
encoding the antigen, e) the nucleic acid encoding the antigen and
at least one other nucleic acid encoding one other antigen, and/or
f) the nucleic acid encoding the antigen and the adjuvant.
[0697] 346. The method of any one of embodiments 215-245, 247-249
and 251-345, wherein the conditioned plurality of modified PBMCs
further comprises an agent that enhances the viability and/or
function of the modified PBMCs as compared to a corresponding
modified PBMCs that does not comprise the agent.
[0698] 347. The method of any one of embodiments 215-245, 247-249
and 251-346, wherein the conditioned plurality of modified PBMCs
further comprises an agent that enhances the viability and/or
function of the modified PBMCs upon freeze-thaw cycle as compared
to a corresponding modified PBMCs that does not comprise the
agent.
[0699] 348. The method of any one of embodiments 215-245, 247-249
and 251-347, wherein at least about 70%, about 80%, or about 90% of
the conditioned plurality of modified PBMCs are viable after up to
1, 2, 3, 4, 5 freeze-thaw cycles.
[0700] 349. The method of embodiment 348, wherein the agent is a
compound that enhances endocytosis, a stabilizing agent or a
co-factor.
[0701] 350. The method of embodiment 349, wherein the agent is
albumin.
[0702] 351. The method of embodiment 350, wherein the albumin is
mouse, bovine, or human albumin.
[0703] 352. The method of 351, wherein the agent is one or more of:
a divalent metal cation, glucose, ATP, potassium, glycerol,
trehalose, D-sucrose, PEG1500, L-arginine, L-glutamine, or
EDTA.
[0704] 353. The method of embodiment 351 or 352, wherein the agent
is one or more of: Sodium pyruvate, adenine, Rejuvesol.RTM.,
trehalose, dextrose, mannose, sucrose, human serum albumin (HSA),
PlasmaLyte.RTM., DMSO, Cryostor.RTM. CS2, Cryostor.RTM. CS5,
Cryostor.RTM. CS10, Cryostor.RTM. CS15, HEPES, glycerol,
glutathione, HypoThermosol.RTM..
[0705] 354. The method of embodiment 353, wherein the agent
comprises mouse serum albumin (MSA).
[0706] 355. The method of embodiment 354, wherein the agent
comprises human serum albumin (HSA).
[0707] 356. The method of any one of embodiments 215-245, 247-249
and 251-355 wherein the cells are further modified to increase
expression of one or more of co-stimulatory molecules.
[0708] 357. The method of embodiment 356, wherein the
co-stimulatory molecule is B7-H2 (ICOSL), B7-1 (CD80), B7-2 (CD86),
CD70, LIGHT, HVEM, CD40, 4-1BBL, OX40L, TL1A, GITRL, CD30L, TIM4,
SLAM, CD48, CD58, CD155, CD112, or scFv anti-CD28.
[0709] 358. The method of embodiment 356, wherein the
co-stimulatory molecule is a Signal 2 effector.
[0710] 359. The method of any one of embodiments 356-358, wherein
the cell comprises a nucleic acid (e.g., mRNA) that results in
increased expression of the one or more co-stimulatory
molecules.
[0711] 360. The method of embodiment 359, wherein the nucleic acid
is an mRNA encoding the co-stimulatory molecule.
[0712] 361. The method of any one of embodiments 215-245, 247-249
and 251-360 wherein the cells are further modified to increase
expression a cytokine.
[0713] 362. The method of embodiment 361, wherein the cytokine is
IL-12, IL-2, IFN-.alpha., or IL-21.
[0714] 363. The method of embodiment 356, wherein the
co-stimulatory molecule is a Signal 3 effector.
[0715] 364. The method of any one of embodiments 361-363, wherein
the cell comprises a nucleic acid (e.g., mRNA) that results in
increased expression of the one or more cytokines.
[0716] 365. The method of embodiment 364, wherein the nucleic acid
is an mRNA encoding the cytokine.
[0717] 366. The method of any one of embodiments 215-245, 247-249
and 251-365, wherein the modified PBMCs comprise a further
modification to modulate MHC class I expression.
[0718] 367. The method of any one of embodiments 215-245, 247-249
and 251-366, wherein the modified PBMCs comprise a further
modification to modulate MHC class II expression.
[0719] 368. The method of embodiment 366, wherein an innate immune
response mounted in an individual in response to administration, in
an allogeneic context, of the modified PBMCs is reduced compared to
an innate immune response mounted in an individual in response to
administration, in an allogeneic context, of corresponding modified
PBMCs that do not comprise the further modification.
[0720] 369. The method of embodiment 366 or 367, wherein the
circulating half-life of the modified PBMCs in an individual to
which they were administered is increased compared to the
circulating half-life of corresponding modified PBMCs that do not
comprise the further modification in an individual to which they
were administered.
[0721] 370. The method of any one of embodiments 215-245, 247-249
and 251-369, wherein the plurality of input PBMCs comprises one or
more of T cell, B cell, NK cell, monocytes, dendritic cells or NK-T
cells.
[0722] 371. The method of any one of embodiments 215-245, 247-249
and 251-370, wherein the plurality of input PBMCs comprises one or
more of CD3+ T cells, CD20+ B cells, CD14+ monocytes, or CD56+NK
cells.
[0723] 372. The method of any one of embodiments 215-245, 247-249
and 251-371, wherein the plurality of input PBMCs comprises T
cells, B cells, NK cells and monocytes, and wherein the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in the plurality of input PBMCs is essentially the same as the
ratio of T cells, B cells, NK cells and monocytes to the total
number of PBMCs in whole blood.
[0724] 373. The method of any one of embodiments 215-245, 247-249
and 251-371, wherein the plurality of input PBMCs comprises T
cells, B cells, NK cells and monocytes, and wherein the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in the plurality of input PBMCs is essentially the same as the
ratio of T cells, B cells, NK cells and monocytes to the total
number of PBMCs in a leukapheresis product from whole blood.
[0725] 374. The method of any one of embodiments 215-245, 247-249
and 251-373, wherein the plurality of input PBMCs comprises T
cells, B cells, NK cells and monocytes, and wherein the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in the plurality of input PBMCs differs by not more than 10% from
the ratio of T cells, B cells, NK cells and monocytes to the total
number of PBMCs in whole blood.
[0726] 375. The method of any one of embodiments 215-245, 247-249
and 251-374, wherein the plurality of input PBMCs comprises T
cells, B cells, NK cells and monocytes, and wherein the ratio of T
cells, B cells, NK cells and monocytes to the total number of PBMCs
in the plurality of input PBMCs differs by not more than 10% from
the ratio of T cells, B cells, NK cells and monocytes to the total
number of PBMCs in whole blood.
[0727] 376. The method of any one of embodiments 215-245, 247-249
and 251-375, wherein:
(a) at least about 25% of the input PBMCs are T cells; (b) at least
about 2.5% of the input PBMCs are B cells; (c) at least about 3.5%
of the input PBMCs are NK cells; or (d) at least about 4% of the
input PBMCs are monocytes.
[0728] 377. The method of any one of embodiments 215-245, 247-249
and 251-376, wherein:
(a) at least about 20% of the modified PBMCs are T cells; (b) at
least about 2% of the modified PBMCs are B cells; (c) at least
about 3% of the modified PBMCs are NK cells; or (d) at least about
3% of the modified PBMCs are monocytes.
[0729] 378. The method of any one of embodiments 215-245, 247-249
and 251-377, wherein:
(a) not more than about 70% of the input PBMCs are T cells; (b) not
more than about 14% of the input PBMCs are B cells; (c) not more
than about 35% of the input PBMCs are NK cells; or (d) not more
than about 25% of the input PBMCs are monocytes.
[0730] 379. The method of any one of embodiments 215-245, 247-249
and 251-378, wherein:
(a) not more than about 80% of the modified PBMCs are T cells; (b)
not more than about 16% of the modified PBMCs are B cells; (c) not
more than about 40% of the modified PBMCs are NK cells; or (d) not
more than about 30% of the modified PBMCs are monocytes.
[0731] 380. The method of any one of embodiments 215-245, 247-249
and 251-379, wherein:
(a) about 25% to about 70% of the modified PBMCs are T cells; (b)
about 2.5% to about 14% of the modified PBMCs are B cells; (c)
about 3.5% to about 35% of the modified PBMCs are NK cells; or (d)
about 4% to about 25% of the modified PBMCs are monocytes.
[0732] 381. The method of any one of embodiments 215-245, 247-249
and 251-380, wherein: (a) the percentage of T cells within the
plurality of modified PBMCs and the percentage of T cells within
the plurality of input PBMCs differ by no more than 10% by number;
(b) the percentage of B cells within the plurality of modified
PBMCs and the percentage of B cells within the plurality of input
PBMCs differ by no more than 10% by number; (c) the percentage of
NK cells within the plurality of modified PBMCs and the percentage
of NK cells within the plurality of input PBMCs differ by no more
than 10% by number; and/or (d) the percentage of monocytes within
the plurality of modified PBMCs and the percentage of monocytes
within the plurality of input PBMCs differ by no more than 10% by
number.
[0733] 382. The method of any one of embodiments 215-245, 247-249
and 251-378, wherein: one or more co-stimulatory molecules is
upregulated in the B cells of the conditioned plurality of modified
PBMCs compared to the B cells in the plurality of input PBMCs,
wherein the co-stimulatory molecule is CD80 or CD86.
[0734] 383. The method of embodiment 382, wherein the CD80 and/or
CD86 is upregulated in the B cells of the conditioned plurality of
modified PBMCs by more than about 1.2-fold, 1.5-fold, 1.8-fold,
2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold
compared to the B cells in a plurality of nonconditioned PBMCs.
[0735] 384. The method of embodiment 382 or 383, wherein the
co-stimulatory molecule is CD86.
[0736] 385. The method of any one of embodiments 382-384, wherein
the conditioned modified PBMCs have increased expression of one or
more of IFN-.gamma., IL-6, MCP-1, MIP-10, IP-10, or TNF-.alpha.
compared to a plurality of unconditioned PBMCs.
[0737] 386. The method of embodiment 385, wherein the expression of
one or more of IFN-.gamma., IL-6, MCP-1, MIP-10, IP-10, or
TNF-.alpha. is increased by about 1.2-fold, 1.5-fold, 1.8-fold,
2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more than 10-fold
compared to the plurality of unconditioned PBMCs.
[0738] 387. The method of any one of embodiments 215-245, 247-249
and 251-385, wherein the modified PBMCs are allogeneic to the
individual.
[0739] 388. The method of any one of embodiments 215-245, 247-249
and 251-385, wherein the modified PBMCs are autologous to the
individual.
[0740] 389. The method of any one of embodiments 215-245, 247-249
and 251-388, wherein the individual is pre-conditioned to modulate
inflammation and/or an immune response.
[0741] 390. The method of any one of embodiments 215-245, 247-249
and 251-389, further comprising administering to the individual a
third adjuvant.
[0742] 391. The method of embodiment 390, wherein the third
adjuvant is IFN-.alpha. or a CpG ODN.
[0743] 392. The method of embodiment 390, wherein the third
adjuvant is CpG 7909.
[0744] 393. The method of any one of embodiments 390-392, wherein
the plurality of modified PBMCs and the third adjuvant are
administered concurrently or simultaneously.
[0745] 394. The method of any one of embodiment 390-392, wherein
the plurality of modified PBMCs and the third adjuvant are
administered sequentially.
[0746] 395. The method of any one of embodiments 390-394, wherein
the plurality of modified PBMCs is administered prior to
administering the third adjuvant.
[0747] 396. The method of any one of embodiments 390-395, wherein
the plurality of modified PBMCs is administered following
administration of the third adjuvant.
[0748] 397. The method of embodiments 215-245, 247-249 and 251-396,
wherein the modified PBMCs is administered prior to, concurrently
with, or following administration of a cytokine.
[0749] 398. The method of embodiment 397, wherein the cytokine is
one or more of: IL-2, IL-7, IL-12a IL-12b, or IL-15.
[0750] 399. The method of embodiments 215-245, 247-249 and 251-398,
wherein the modified PBMCs is administered prior to, concurrently
with, or following administration of an immune checkpoint
inhibitor.
[0751] 400. The method of embodiment 399, wherein the immune
checkpoint inhibitor is targeted to any one of PD-1, PD-L1, CTLA-4,
LAG3, VISTA, and TIM-3.
[0752] 401. The method of embodiment 400, wherein the immune
checkpoint inhibitor is targeted to PD-1.
[0753] 402. The method of embodiment 400, wherein the immune
checkpoint inhibitor is targeted to PD-L1.
[0754] 403. The method of embodiments 215-245, 247-249 and 251-402,
wherein the modified PBMCs is administered prior to, concurrently
with, or following administration of a therapeutic agent.
[0755] 404. The method of embodiment 403, wherein the therapeutic
agent is a chemotherapeutic agent.
[0756] 405. The method of any one of embodiments 215-245, 247-249
and 251-404, wherein administration of the modified PBMCs to the
individual results in activation and/or expansion of cytotoxic T
lymphocytes (CTLs) specific for the antigen.
[0757] 406. The method of any one of embodiments 215-245, 247-249
and 251-405 wherein administration of the modified PBMCs to the
individual results in activation and/or expansion of helper T (Th)
cells specific for the antigen.
[0758] 407. The method of any one of embodiments 215-245, 247-249
and 251-406, wherein the amount of the modified PBMCs administered
to the individual is between about 1.times.10.sup.4 and about
1.times.10.sup.12 cells.
[0759] 408. The method of embodiment 407, wherein the amount of the
modified PBMCs administered to the individual is between about
1.times.10.sup.5 and about 1.times.10.sup.12 cells.
[0760] 409. The method of embodiment 407 or 408, wherein the amount
of the modified PBMCs administered to the individual is between
about be 5.times.10.sup.5 and about 2.5.times.10.sup.6 cells/kg
body weight.
[0761] 410. The method of any one of embodiments 215-245, 247-249
and 251-409, wherein the method comprises multiple administrations
of the modified PBMCs.
[0762] 411. The method of embodiment 410, wherein the method
comprises about 3 to about 9 administrations.
[0763] 412. The method of embodiment 410 or 411, wherein the time
interval between two successive administrations of the plurality of
modified PBMCs is between about 1 day and about 30 days.
[0764] 413. The method of any one of embodiments 410-412, wherein
the time interval between two successive administrations of the
plurality of modified PBMCs is about 21 days.
[0765] 414. The method of any one of embodiments 215-245, 247-249
and 251-413, wherein the individual is positive for expression of
HLA-A2.
[0766] 415. The method of any one of embodiments 215-245, 247-249
and 251-414, wherein at least one cell in the plurality of modified
PBMCs is positive for expression of HLA-A2.
[0767] 416. A method for generating a conditioned plurality of
modified PBMCs comprising a human papillomavirus (HPV) antigen,
comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, thereby generating the conditioned plurality of
modified PBMCs comprising the HPV antigen.
[0768] 417. A method for generating a conditioned plurality of
modified PBMCs comprising an HPV antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, wherein the CpG ODN is CpG 7909, thereby generating
the conditioned plurality of modified PBMCs comprising the HPV
antigen.
[0769] 418. A method for generating a conditioned plurality of
modified PBMCs comprising an HPV antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for
about 1 hour to about 24 hours for the modified PBMCs comprising
the HPV antigen to condition, thereby generating the conditioned
plurality of modified PBMCs comprising the HPV antigen.
[0770] 419. A method for generating a conditioned plurality of
modified PBMCs comprising an HPV antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; and c) incubating the plurality
of modified PBMCs comprising the HPV antigen with a CpG ODN for
about 1 hour to about 24 hours for the modified PBMCs comprising
the HPV antigen to condition, wherein the CpG ODN is CpG 7909,
thereby generating the conditioned plurality of modified PBMCs
comprising the HPV antigen.
[0771] 420. The method of any one of embodiments 416-419, wherein
the diameter of the constriction is about 4 .mu.m to about 10
.mu.m.
[0772] 421. The method of any one of embodiments 416-420, wherein
the diameter of the constriction is about 3 .mu.m to about 6
.mu.m.
[0773] 422. The method of any one of embodiments 416-421, wherein
the diameter of the constriction is (a) about 4.2 .mu.m to about 6
.mu.m; or (b) about 4.5 .mu.m.
[0774] 423. The method of any one of embodiments 416-422, wherein
the plurality of modified PBMCs comprising the HPV antigen is
incubated with a CpG ODN for (a) about 2 hour to about 10 hours;
(b) about 3 hours to about 6 hours; or (c) about 4 hours.
[0775] 424. A method for generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for a sufficient time
for the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen.
[0776] 425. A method for generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for a sufficient time
for the modified PBMCs comprising the antigen to condition, wherein
the CpG ODN is CpG 7909, thereby generating the conditioned
plurality of modified PBMCs comprising the antigen.
[0777] 426. A method for generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for about 1 hour to
about 24 hours for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen.
[0778] 427. A method for generating a conditioned plurality of
modified PBMCs comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; and c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for about 1 hour to
about 24 hours for the modified PBMCs comprising the antigen to
condition, wherein the CpG ODN is CpG 7909, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0779] 428. The method of any one of embodiments 424-427, wherein
the diameter of the constriction is about 4 .mu.m to about 10
.mu.m.
[0780] 429. The method of any one of embodiments 424-428, wherein
the diameter of the constriction is about 3 .mu.m to about 6
.mu.m.
[0781] 430. The method of any one of embodiments 424-429, wherein
the diameter of the constriction is (a) about 4.2 .mu.m to about 6
.mu.m; or (b) about 4.5 .mu.m.
[0782] 431. The method of any one of embodiments 424-430, wherein
the plurality of modified PBMCs comprising the HPV antigen is
incubated with a CpG ODN for (a) about 2 hours to about 10 hours;
(b) about 3 hours to about 6 hours; or (c) about 4 hours.
[0783] 432. A method for stimulating an immune response against an
HPV antigen in an individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, thereby generating the conditioned plurality of
modified PBMCs comprising the HPV antigen; and d) administering the
conditioned plurality of modified PBMCs comprising the HPV antigen
to the individual.
[0784] 433. A method for stimulating an immune response against an
HPV antigen in an individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for a
sufficient time for the modified PBMCs comprising the HPV antigen
to condition, wherein the CpG ODN is CpG 7909, thereby generating
the conditioned plurality of modified PBMCs comprising the HPV
antigen; and d) administering the conditioned plurality of modified
PBMCs comprising the HPV antigen to the individual.
[0785] 434. A method for stimulating an immune response against an
HPV antigen in an individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for about
1 hour to about 24 hours for the modified PBMCs comprising the HPV
antigen to condition, thereby generating the conditioned plurality
of modified PBMCs comprising the HPV antigen; and d) administering
the conditioned plurality of modified PBMCs comprising the HPV
antigen to the individual.
[0786] 435. A method for stimulating an immune response against an
HPV antigen in an individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for the HPV antigen
to pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the HPV
antigen for a sufficient time to allow the HPV antigen to enter the
perturbed input PBMCs, thereby generating a plurality of modified
PBMCs comprising the HPV antigen; c) incubating the plurality of
modified PBMCs comprising the HPV antigen with a CpG ODN for about
1 hour to about 24 hours for the modified PBMCs comprising the HPV
antigen to condition, wherein the CpG ODN is CpG 7909, thereby
generating the conditioned plurality of modified PBMCs comprising
the HPV antigen; and d) administering the conditioned plurality of
modified PBMCs comprising the HPV antigen to the individual.
[0787] 436. The method of any one of embodiments 432-435, wherein
the diameter of the constriction is about 4 .mu.m to about 10
.mu.m.
[0788] 437. The method of any one of embodiments 432-436, wherein
the diameter of the constriction is about 3 .mu.m to about 6
.mu.m.
[0789] 438. The method of any one of embodiments 432-437, wherein
the diameter of the constriction is (a) about 4.2 .mu.m to about 6
.mu.m; or (b) about 4.5 .mu.m.
[0790] 439. The method of any one of embodiments 432-438, wherein
the plurality of modified PBMCs comprising the HPV antigen is
incubated with a CpG ODN for (a) about 2 hours to about 10 hours;
(b) about 3 hours to about 6 hours; or (c) about 4 hours.
[0791] 440. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for an antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for a sufficient time
for the modified PBMCs comprising the antigen to condition, thereby
generating the conditioned plurality of modified PBMCs comprising
the antigen; and d) administering the conditioned plurality of
modified PBMCs comprising the antigen to the individual.
[0792] 441. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for an antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for a sufficient time
for the modified PBMCs comprising the antigen to condition, wherein
the CpG ODN is CpG 7909, thereby generating the conditioned
plurality of modified PBMCs comprising the antigen; and d)
administering the conditioned plurality of modified PBMCs
comprising the antigen to the individual.
[0793] 442. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for an antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for about 1 hour to
about 24 hours for the modified PBMCs comprising the antigen to
condition, thereby generating the conditioned plurality of modified
PBMCs comprising the antigen; and d) administering the conditioned
plurality of modified PBMCs comprising the antigen to the
individual.
[0794] 443. A method for stimulating an immune response in an
individual, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is about 3 .mu.m to about 10 .mu.m, thereby causing
perturbations of the input PBMCs large enough for an antigen to
pass through to form a plurality of perturbed input PBMCs; b)
incubating the plurality of perturbed input PBMCs with the antigen
for a sufficient time to allow the antigen to enter the perturbed
input PBMCs, thereby generating a plurality of modified PBMCs
comprising the antigen; c) incubating the plurality of modified
PBMCs comprising the antigen with a CpG ODN for about 1 hour to
about 24 hours for the modified PBMCs comprising the antigen to
condition, wherein the CpG ODN is CpG 7909, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen; and
d) administering the conditioned plurality of modified PBMCs
comprising the antigen to the individual.
[0795] 444. The method of any one of embodiments 440-443, wherein
the diameter of the constriction is about 4 .mu.m to about 10
.mu.m.
[0796] 445. The method of any one of embodiments 440-444, wherein
the diameter of the constriction is about 3 .mu.m to about 6
.mu.m.
[0797] 446. The method of any one of embodiments 440-445, wherein
the diameter of the constriction is (a) about 4.2 .mu.m to about 6
.mu.m; or (b) about 4.5 .mu.m.
[0798] 447. The method of any one of embodiments 440-446, wherein
the plurality of modified PBMCs comprising the HPV antigen is
incubated with a CpG ODN for (a) about 2 hours to about 10 hours;
(b) about 3 hours to about 6 hours; or (c) about 4 hours.
[0799] 448. The method of any one of embodiments 432-447, further
comprising administering to the individual a second adjuvant.
[0800] 449. The method of embodiment 448, wherein the second
adjuvant is IFN-.alpha. or a CpG ODN.
[0801] 450. The method of embodiment 449, wherein the second
adjuvant is CpG 7909.
[0802] 451. The method of any one of embodiments 432-450, wherein
the conditioned plurality of modified PBMCs further comprises an
agent that enhances the viability and/or function of the modified
PBMCs, optionally wherein the agent is one or more of: Sodium
pyruvate, adenine, Rejuvesol.RTM., trehalose, dextrose, mannose,
sucrose, human serum albumin (HSA), PlasmaLyte.RTM., DMSO,
Cryostor.RTM. CS2, Cryostor.RTM. CS5, Cryostor.RTM. CS10,
Cryostor.RTM. CS15, HEPES, glycerol, glutathione,
HypoThermosol.RTM..
[0803] 452. The method of embodiments 432-451, wherein the modified
PBMCs is administered prior to, concurrently with, or following
administration of an immune checkpoint inhibitor.
[0804] 453. The method of embodiment 452, wherein the immune
checkpoint inhibitor is targeted to any one of PD-1, PD-L1, CTLA-4,
LAG3, VISTA, and TIM-3.
[0805] 454. The method of embodiment 453, wherein the immune
checkpoint inhibitor is targeted to PD-1.
[0806] 455. The method of embodiment 453, wherein the immune
checkpoint inhibitor is targeted to PD-L1.
[0807] 456. The method of embodiments 440-455, wherein the modified
PBMCs is administered prior to, concurrently with, or following
administration of a therapeutic agent.
[0808] 457. The method of embodiment 456, wherein the therapeutic
agent is a chemotherapeutic agent.
Additional Embodiments
[0809] 1. A plurality of modified peripheral blood mononuclear
cells (PMBCs) comprising an antigen, wherein the antigen is
exogenous to the modified PBMCs and wherein the plurality of
modified PBMCs comprises two or more of T cells, B cells, NK cells
or monocytes, in particular wherein the antigen is a cancer
antigen, an infectious disease antigen or a viral-disease
associated antigen.
[0810] 2. The plurality of modified PBMCs of embodiment 1, wherein
the antigen is present in at least about 70% of the cells in the
plurality of PBMCs.
[0811] 3. The plurality of modified PBMCs according to embodiments
1 or 2, which is a conditioned plurality of modified PBMCs, in
particular wherein the modified PBMCs comprise an adjuvant.
[0812] 4. The plurality of modified PBMCs of embodiment 3, wherein
the antigen is present in the cytosol and the adjuvant is present
in a vesicle of a cell in the plurality of the modified PBMCs.
[0813] 5. The plurality of modified PBMCs of any one of embodiments
3 or 4, wherein CD80 and/or CD86 is unregulated in the B cells of
the plurality of conditioned PBMCs by more than about 1.2-fold,
1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more
than 10-fold compared to the B cells in a plurality of
unconditioned PBMCs.
[0814] 6. The plurality of modified PBMCs of any one of embodiments
3-5, wherein the expression of one or more of IFN-.gamma., IL-6,
MCP-1, MIP-1.beta., IP-10, or TNF-.alpha. is increased in the PBMCs
of the plurality of conditioned PBMCs by more than about 1.2-fold,
1.5-fold, 1.8-fold, 2-fold, 3-fold, 4-fold, 5-fold, 8-fold, or more
than 10-fold compared to the plurality of unconditioned PBMCs.
[0815] 7. The plurality of modified PMBCs according to any one of
embodiments 1-6, prepared by a process comprising the steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen.
[0816] 8. The conditioned plurality of modified PBMCs according to
any one of embodiments 3-7, prepared by a process comprising the
steps of:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and c)
incubating the plurality of modified PBMCs comprising the antigen
with the adjuvant for a sufficient time for the modified PBMCs
comprising the antigen to condition, thereby generating the
conditioned plurality of modified PBMCs comprising the antigen.
[0817] 9. The plurality of modified PBMCs of any one of embodiments
1-8, wherein:
(a) the percentage of T cells within the plurality of modified
PBMCs and the percentage of T cells within the plurality of input
PBMCs differ by no more than about 10%; (b) the percentage of B
cells within the plurality of modified PBMCs and the percentage of
B cells within the plurality of input PBMCs differ by no more than
about 10%; (c) the percentage of NK cells within the plurality of
modified PBMCs and the percentage of NK cells within the plurality
of input PBMCs differ by no more than about 10%; and/or (d) the
percentage of monocytes within the plurality of modified PBMCs and
the percentage of monocytes within the plurality of input PBMCs
differ by no more than about 10%.
[0818] 10. A method for generating a plurality of modified PBMCs
comprising an antigen, comprising:
a) passing a cell suspension comprising a plurality of input PBMCs
through a cell-deforming constriction, wherein a diameter of the
constriction is a function of a diameter of the input PBMCs in the
suspension, thereby causing perturbations of the input PBMCs large
enough for the antigen to pass through to form a plurality of
perturbed input PBMCs; b) incubating the plurality of perturbed
input PBMCs with the antigen for a sufficient time to allow the
antigen to enter the perturbed input PBMCs, thereby generating a
plurality of modified PBMCs comprising the antigen; and optionally
c) incubating the plurality of modified PBMCs comprising the
antigen with an adjuvant for a sufficient time for the modified
PBMCs comprising the antigen to condition, thereby generating a
conditioned plurality of modified PBMCs comprising the antigen.
[0819] 11. The plurality of modified or conditioned PBMCs of any
one of embodiments 7-9 or the method of embodiment 10, wherein the
diameter of the constriction is about 60% to about 90% of the mean
diameter of a subpopulation of cells having the smallest diameter
within the plurality of input PBMCs, and/or wherein the diameter of
the constriction is about 20% to about 30% of the mean diameter of
a subpopulation of cells having the largest diameter within the
plurality of input PBMCs.
[0820] 12. The plurality of modified or conditioned PBMCs of any
one of embodiments 7-9 or 11, or the method of embodiment 10 or 11,
wherein the diameter of the constriction is about 3 .mu.m to about
10 .mu.m, in particular about 3 .mu.m to about 6 .mu.m.
[0821] 13. The conditioned plurality of modified PBMCs of any one
of embodiments 8-9 or 11-12, or the method of any one of
embodiments 10-12, wherein the plurality of modified PBMCs is
incubated with the adjuvant for about 1 to about 24 hours for the
modified PBMCs to condition, in particular for about 2 to about 10
hours.
[0822] 14. The conditioned plurality of modified or conditioned
PBMCs of any one of embodiments 3-9 or 11-13, or the method of any
one of embodiments 10-13, wherein the adjuvant is a CpG
oligodeoxynucleotide (ODN), LPS, IFN-.alpha., STING agonists, RIG-I
agonists, poly I:C, R837, R848, a TLR3 agonist, a TLR4 agonist or a
TLR 9 agonist.
[0823] 15. The plurality of modified or conditioned PBMCs of any
one of embodiments 1-9 or 11-14, or the method of any one of
embodiments 10-14, wherein the antigen is a human papillomavirus
(HPV) antigen.
EXAMPLES
[0824] Those skilled in the art will recognize that several
embodiments are possible within the scope and spirit of this
invention. The invention will now be described in greater detail by
reference to the following non-limiting examples. The following
examples further illustrate the invention but, of course, should
not be construed as in any way limiting its scope.
Example 1
[0825] To investigate the efficacy of a conditioned plurality of
PBMCs comprising an antigen in stimulating an immune response in an
individual, a conditioned plurality of PBMCs comprising an
HPV-derived antigen is generated and administered to individuals.
The efficacy of conditioned PBMCs comprising a disease antigen for
use as a therapeutic vaccine is studied, as a monotherapy or when
combined with an additional therapeutic agent, such as a PD-L1
inhibitor and/or a chemotherapeutic agent.
[0826] PBMC-HPV drug substance consists of autologous PBMCs
presenting HLA-A02 restricted E6 and E7 epitopes of HPV16 on MHC-I.
The majority of PBMCs (>90%) consist of T cells, monocytes, NK
cells, and B cells. An illustration of the structure of PBMC-HPV is
presented in FIG. 1A, with the indicated lines representing
full-length E6 and E7 synthetic long peptides (SLPs) that contain
immunogenic epitopes of HPV16. The minimal epitope marked in red
and green, respectively. Once delivered, SLPs are processed to
generate the minimal epitopes, which are subsequently presented on
the MHC-I.
[0827] The cell in the FIG. 1A represents any of the PBMC-HPV cell
types (T cells, monocytes, NK cells, and B cells).
[0828] E6 SLP and E7 SLP used as starting materials in the
production of the PBMC-HPV drug substance are shown below. These
peptides contain antigenic epitopes for HPV16 (shown in bold
letters).
TABLE-US-00001 E6 SLP: (SEQ ID NO: 19) QLCTELQTTIHDIILECVYCKQQLL E7
SLP: (SEQ ID NO: 23) QLCTELQTYMLDLQPETTYCKQQLL
[0829] After being delivered into the cell cytosol during the
manufacturing process, these peptides are processed by the cells
and the resultant segments containing antigenic epitopes are
presented on the MHC-I of the various PBMC cells. The E6 SLP is a
25 amino acid peptide from the native E6 protein that was chosen
because it contains the HLA-A2 antigenic peptide TIHDIILECV (SEQ ID
NO: 1), which has been cited as a HLA-A2 restricted immunodominant
epitope in the E6 protein. The E7 SLP contains the HLA-A2
restricted immunodominant epitope YMLDLQPETT (SEQ ID NO: 3) within
flanking amino acids from the E6 protein. This E7 epitope has been
widely cited as an antigenic E7 peptide. The E7 epitope in this
flanking sequence, which is not identical to the native E7 protein,
was found to be more efficiently processed and presented in vitro
by human antigen presenting cells than an SLP containing the native
E7 flanking sequence.
[0830] As part of the PBMC-HPV drug substance manufacturing
process, the PBMC-HPV cells are conditioned with CpG 7909, a CpG
oligodeoxyribonucleotide (ODN) that stimulates toll-like receptor
(TLR9) signaling. This maturation results in production of
inflammatory cytokines (e.g. IL-6) by PBMC-HPV cells and
upregulation of costimulatory molecules (e.g. CD86) and MHC-I on B
cells. After the maturation period, the PBMC-HPV drug substance is
washed to remove CpG 7909 and accumulated cytokines, and
subsequently formulated into a composition comprising a conditioned
plurality of PBMCs comprising an HPV antigen, called the
SQZ-PBMC-HPV drug product.
[0831] The PBMC-HPV drug substance consists of the cells with E6
and E7 SLPs delivered to the cytosol and after CpG 7909 matures the
cells, but immediately prior to washing away the CpG 7909, and the
formulation and filling required to generate the SQZ-PBMC-HPV drug
product.
[0832] To manufacture the drug product, the PBMC-HPV drug substance
is washed twice and subsequently formulated in a solution. For
example, the solution can contain containing 50% (v/v) of a
cryogenic preservation media (such as CryoStor.RTM. CS10), 30%
(v/v) of a hypothermic preservation media (such as
HypoThermosol.RTM. FRS), and 20% (v/v) of albumin (such as
Albuked.TM. 25 (25% Human Serum Albumin); NDC #76125-0792-10)
[0833] Approximately 6-8 days after leukapheresis, patients receive
the SQZ-PBMC-HPV drug product intravenously (IV). The dose of the
SQZ PBMC-HPV drug product varies according to the patient's dose
cohort and is dosed on live cells/kg basis.
[0834] The first-in-human (FIH) study consists of an Escalation
Phase and an Expansion Phase for SQZ-PBMC-HPV and
SQZ-PBMC-HPV+atezolizumab
[0835] The SQZ-PBMC-HPV Escalation Phase comprises the following
cohorts: [0836] (1) a low cell dose cohort with an initial
administration followed by two boosters of SQZ-PBMC-HPV, one 3
weeks and one 6 weeks after the initial dose, [0837] (2) a low cell
dose initial administration followed by 5 boosters of SQZ-PBMC-HPV,
administered in 3-week intervals, [0838] (3) a high cell dose
cohort with an initial administration followed by two boosters of
SQZ-PBMC-HPV, one 3 weeks and one 6 weeks after the initial dose of
3 equal aliquots, [0839] (4) a high cell dose cohort with an
initial administration followed by two boosters of SQZ-PBMC-HPV,
one 3 weeks and one 6 weeks after the initial dose. In this cohort,
CpG 7909 will be co-administered each time following
SQZ-PBMC-HPV.
[0840] While the main focus of the FIH study is be the evaluation
of the administration of SQZ-PBMC-HPV, the study includes the
evaluation of the co-administration of SQZ-PBMC-HPV and CpG 7909 in
one cohort (Cohort 4). Cohort 4 provides information whether or not
CpG7909 is CO-administered for subsequent cohorts and the Expansion
Phase. SQZ-PBMC-HPV is administered by IV first, followed by IV
administration of CpG 7909 in Cohort 4.
[0841] Following the analysis of at least 4 patients in each cohort
regarding safety, tolerability and the impact of co-administration
of CpG 7909, a decision is made together with the study
investigators whether the drug product SQZ-PBMC-HPV is
co-administered with CpG 7909 or not in the Escalation Phase for
SQZ-PBMC-HPV+atezolizumab. The following cohorts are tested: [0842]
(5) a low cell dose cohort+atezolizumab with an initial
administration followed by two boosters of
SQZ-PBMC-HPV+atezolizumab, one 3 weeks and one 6 weeks after the
initial dose, [0843] (6) a low cell dose cohort+atezolizumab with
initial administration followed by at least 5 boosters (max. 9
dependent on number of harvested cells) of
SQZ-PBMC-HPV+atezolizumab, administered in 3-week intervals, [0844]
(7) a high cell dose cohort+atezolizumab with an initial
administration followed by two boosters of
SQZ-PBMC-HPV+atezolizumab, one 3 weeks and one 6 weeks after the
initial dose.
[0845] FIG. 1B and FIG. 1C show representative schematics of cohort
treatments for the SQZ-PBMC-HPV monotherapy and
SQZ-PBMC-HPV+atezolizumab combination therapy, respectively
[0846] For an Expansion Phase study, up to 3 baskets for dosing
SQZ-PMBC-HPV as a monotherapy are: [0847] (1) HPV16-positive Head
& Neck Squamous Cell Cancer at the selected Recommended Phase 2
Dose (RP2D) Regimen, [0848] (2) HPV16-positive Cervical Cancer at
the selected Recommended Phase 2 Dose (RP2D) Regimen, [0849] (3)
Other HPV16-positive cancer at the selected Recommended Phase 2
Dose (RP2D) Regimen,
[0850] Up to 3 baskets for SQZ-PBMC-HPV+atezolizumab are:
[0851] HPV16-positive Head and Neck Squamous Cell Cancer at the
selected Recommended Phase 2 Dose (RP2D) regimen for
SQZ-PBMC-HPV,
[0852] HPV16-positive Cervical Cancer at the selected Recommended
Phase 2 Dose (RP2D) regimen for SQZ-PBMC-HPV,
[0853] Other HPV16-positive cancer at the selected Recommended
Phase 2 Dose (RP2D) regimen for SQZ-PBMC-HPV
Example 2
[0854] In order to quantify SQZ-mediated delivery to individual
immune cell subsets, mouse splenocytes were loaded with a
fluorescent tracer molecule and assessed for viability and
delivery.
Method
[0855] Splenocytes were isolated from C57BL/6J female mice (20
M/mL) and loaded using SQZ (30, 60 and 90 psi; 4 .mu.m
constriction) with 100 .mu.g/mL of fluorescently-labeled dextran (3
kDa) in RPMI and the viability and percent delivery of dextran to
individual immune cell subsets within the mixed splenocyte
population by flow cytometry.
Results
[0856] As shown in FIG. 2, the percent delivery of dextran to
splenocytes increased significantly (P<0.001; each condition
relative to Endo) with increasing pressure, with only a slight
decrease in viability with the 30 and 60 psi conditions, and a
larger decrease in viability with the 90 psi condition. B and NK
cells had the highest levels of delivery amongst the other cell
populations, although there was still appreciable amount of
delivery in T cells and monocytes. At the condition that gave the
highest delivery with only minimal impact on viability (60 psi),
all cell subsets had a percent delivery within .about.15% (60-75%
delivery across all cell types). These data show that SQZ can
efficiently deliver molecules to each immune cell subset in a mixed
population of murine splenocytes simultaneously, with little impact
on viability.
Example 3
[0857] In order to evaluate the impact of different adjuvant
strategies, mixed splenocytes and isolated B cells were loaded with
a model antigen and conditioned and/or co-injected with adjuvant
and the relative percentage of inflammatory cytokine
IFN-.gamma.+CD8+ T cells was measured by flow cytometry.
[0858] Method
[0859] At Day 0, splenocytes were obtained from spleens of female
C57BL/6J donor mice, along with B cells isolated from the
splenocytes for one group via immunomagnetic separation, loaded
with Ova protein (400 .mu.g/mL) by SQZ (60 psi; 4 .mu.m
constriction) and incubated in either media (R10) alone or media
with CpG 1826 (1 .mu.M) for 16 h. Female C57BL/6J recipient mice
(5/group) were injected retro-orbitally on Day 1 with 100 .mu.L of
either vehicle (PBS), B cells (5.times.10.sup.6 cells/mL),
splenocytes (5.times.10.sup.6 cells/mL) or splenocytes co-injected
with 25 .mu.g CpG1826. On Day 8, spleens were harvested,
restimulated with SIINFEKL (SEQ ID NO: 54) (1 .mu.g/mL) and the
percentage of IFN-.gamma.+CD8+ T cells was determined by
intracellular cytokine staining (ICS).
Results
[0860] As shown in FIG. 3, the percentage of IFN-.gamma.+CD8+ T
cells for mice treated with Ova-loaded splenocytes was
significantly higher when splenocytes were conditioned with CpG for
16 h (P<0.005 relative to all other conditions; Data represents
3 independent experiments); however, there was no significant
increase in % IFN-.gamma.+CD8+ T cells when mice were co-injected
with CpG or treated with CpG-matured B cells (B.sub.APC). These
data show that treatment with CpG-matured, Ova-loaded splenocytes
lead to a significant increase in inflammatory cytokine production
in antigen-specific CD8+ T cells, while Ova-loaded B cells, or
splenocytes co-injected with CpG, did not induce an appreciable
response.
Example 4
[0861] In order to determine if antigen-loaded splenocytes
co-injected with lower doses of CpG can elicit an antigen-specific
response, splenocytes were loaded with a model antigen and either
matured with CpG, co-injected with CpG or matured and co-injected
with increasing doses of CpG and the relative percentage of
inflammatory cytokine IFN-.gamma.+CD8+ T cells was measured by flow
cytometry.
Methods
[0862] At Day 0, splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. These mixed splenocytes were then loaded with Ova protein
(400 .mu.g/mL) by SQZ (60 psi; 4 .mu.m constriction) and incubated
in either media (R10) alone or media with CpG 1826 (1 .mu.M) for 16
h. Female C57BL/6J recipient mice (5/group) were injected
retro-orbitally on Day 1 with 100 .mu.L of either vehicle (PBS),
splenocytes (1.times.10.sup.6 cells/mouse) matured with CpG,
splenocytes co-injected with 25 .mu.g CpG1826 or splenocytes
matured with CpG that are co-injected with different doses of CpG
(0.1-10 .mu.g). On Day 8, spleens were harvested, restimulated with
SIINFEKL (SEQ ID NO: 54) (1 .mu.g/mL) and the percentage of
IFN-.gamma.+CD8+ T cells was determined by intracellular cytokine
staining (ICS).
Results
[0863] As shown in FIG. 4, The percentage of IFN-.gamma.+CD8+ T
cells for mice treated with Ova-loaded splenocytes was
significantly higher when splenocytes were matured with CpG for 16
h (P<0.05 relative to untreated); however, there was no
significant increase in % IFN-.gamma.+CD8+ T cells when mice were
co-injected with CpG relative to control. Additionally, there was
no statistically significant increase in the percentage of %
IFN-.gamma.+CD8+ T cells when splenocytes were matured with CpG
compared to matured and co-injected with any dose of CpG, with an
apparent slight decrease with the 5 .mu.g CpG co-injection. These
data show that co-injection of CpG combined with CpG-matured,
Ova-loaded splenocytes does not lead to a significant increase in
inflammatory cytokine production in antigen-specific CD8+ T
cells.
Example 5
[0864] In order to determine if antigen-loaded splenocytes
co-injected with different adjuvants can elicit an antigen-specific
response, splenocytes were loaded with a model antigen and either
matured with CpG, co-injected with CpG or matured with CpG and
co-injected with either CpG or IFN-.alpha. and the relative
percentage of inflammatory cytokine IFN-.gamma.+CD8+ T cells was
measured by flow cytometry.
Methods
[0865] At Day 0, splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. These mixed splenocytes were then loaded with Ova protein
(400 .mu.g/mL) by SQZ (60 psi; 4 .mu.m constriction) and incubated
in either media (R10) alone or media with CpG 1826 (1 .mu.M) for 16
h. Female C57BL/6J recipient mice (5/group) were injected
retro-orbitally on Day 1 with 100 .mu.L of either vehicle (PBS),
splenocytes (1.times.10.sup.6 cells/mouse) matured with CpG,
splenocytes co-injected with 1 .mu.g CpG1826 or splenocytes matured
with CpG that are co-injected with either CpG or 10000 U
IFN-.alpha.. On Day 8, spleens were harvested, restimulated with
SIINFEKL (SEQ ID NO: 54) (1 .mu.g/mL) and the percentage of
IFN-.gamma.+CD8+ T cells was determined by intracellular cytokine
staining (ICS).
Results
[0866] As shown in FIG. 5, the percentage of IFN-.gamma.+CD8+ T
cells for mice treated with Ova-loaded splenocytes was highest when
splenocytes were matured with CpG for 16 h, with a significant
increase when compared to untreated (P<0.0001) as well as
co-injected CpG (P<0.005). There was no significant benefit to
maturing splenocytes with CpG and co-injecting them with either
adjuvant, with a trend towards the co-injected adjuvant blunting
the effect. These data show that co-injection of CpG or IFN-.alpha.
combined with CpG-matured, Ova-loaded splenocytes does not lead to
a significant increase in inflammatory cytokine production in
antigen-specific CD8+ T cells.
Example 6
[0867] In order to determine if boosting an antigen-specific
splenocyte vaccine can elicit a greater antigen-specific response,
splenocytes were loaded with a model antigen, matured with CpG, and
injected into recipient mice once (Prime) or twice (Prime-Boost),
with the relative percentage of inflammatory cytokine
IFN-.gamma.+CD8+ T cells measured by flow cytometry.
Methods
[0868] At Day -7, splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. These mixed splenocytes were then loaded with Ova protein
(400 .mu.g/mL) by SQZ (60 psi; 4 .mu.m constriction) and with CpG
1826 (1 .mu.M in R10) for 4 h. Female C57BL/6J recipient mice
(5/group) were injected retro-orbitally on Day -7 and/or Day 0 with
100 .mu.L of splenocytes (1.times.10.sup.5-6 cells/mouse). On Day
7, spleens were harvested, restimulated with SIINFEKL (SEQ ID NO:
54) (1 .mu.g/mL) and the percentage of IFN-.gamma.+CD8+ T cells was
determined by intracellular cytokine staining (ICS).
[0869] Results
[0870] As shown in FIG. 6, while increasing splenocyte dose did
lead to a modest increase in the percentage of IFN-.gamma.+CD8+ T
cells, the addition of a boost after 7 days led to a significant
(P<0.05) increase in IFN-.gamma.+ cells for all of the
splenocyte doses tested. Interestingly, the boost enhancement was
most pronounced with lower cell doses, with the 0.1M dose
exhibiting an 8-fold increase in antigen-specific response. These
data show that, for all doses tested, the use of a boost 7 days
after priming leads to a significant enhancement in the
antigen-specific CD8+ T cell response.
Example 7
[0871] In order to evaluate the importance of cell dose and
relative efficacy of a B cell versus a mixed splenocyte vaccine,
cells were loaded with a model antigen and matured with adjuvant
and the relative percentage of inflammatory cytokine
IFN-.gamma.+CD8+ T cells was measured by flow cytometry.
Methods
[0872] At Day 0, splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. Additionally, isolated B cells were obtained from spleens of
female donor mice via positive immunomagnetic separation. These
different cell compositions were loaded with Ova protein (400
.mu.g/mL) by SQZ (60 psi; 4 .mu.m constriction) and incubated in
either media (R10) alone or media with CpG 1826 (1 .mu.M) for 16 h.
Female C57BL/6J recipient mice (5/group) were injected
retro-orbitally on Day 1 with 100 .mu.L of either vehicle (PBS), B
cells or splenocytes (0.25-4.times.10.sup.6 cells/mL). On Day 8,
spleens were harvested, restimulated with SIINFEKL (SEQ ID NO: 54)
(1 .mu.g/mL) and the percentage of IFN-.gamma.+CD8+ T cells was
determined by intracellular cytokine staining (ICS).
Results
[0873] As shown in FIG. 7, The percentage of IFN-.gamma.+CD8+ T
cells for mice treated with Ova-loaded B cells or splenocytes
exhibited a positive dose-response, with the 4M cell dose leading
to the highest response for both cell types tested. Generally, all
doses of splenocytes led to higher average responses then their
BAPC counterparts, trending towards significance. This data show
that higher cell numbers trend with increased responses and that
splenocytes may induce a higher antigen-specific response.
Example 8
[0874] In order to evaluate the impact of CpG maturation time on
the relative efficacy of mixed splenocytes to induce an
antigen-specific response, splenocytes were loaded with a model
antigen and matured with CpG for varying times and the relative
percentage of inflammatory cytokine IFN-.gamma.+CD8+ T cells was
measured by flow cytometry.
Methods
[0875] At Day 0, splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. Splenocyte were loaded with Ova protein (400 .mu.g/mL) by
SQZ (60 psi; 4 .mu.m constriction) and incubated in either media
(R10) alone or media with CpG 1826 (1 .mu.M) for varying times.
Female C57BL/6J recipient mice (5/group) were injected
retro-orbitally on Day 1 with 100 .mu.L of either vehicle (PBS) or
splenocytes (1.times.10.sup.6 cells/mL). On Day 8, spleens were
harvested, restimulated with SIINFEKL (SEQ ID NO: 54) (1 .mu.g/mL)
and the percentage of IFN-.gamma.+CD8+ T cells was determined by
intracellular cytokine staining (ICS).
Results
[0876] As shown in FIG. 8, The percentage of IFN-.gamma.+CD8+ T
cells for mice treated with Ova-loaded splenocytes increased with
longer CpG maturation times (*P<0.05, **P<0.01, # P<0.005,
all comparisons made to No CpG). There was a significant increase
in the response observed with all maturation times of at least 4
hours. These data show that at least 4 hours CpG maturation time
post-SQZ is necessary to induce a significant antigen-specific
response.
Example 9
[0877] In order to determine the minimum effective cell dose of
splenocytes needed to lead to tumor growth inhibition in a
therapeutic setting, four different doses of splenocytes were
tested in the HPV E7-expressing TC1 tumor model, with the area of
the tumors plotted against time. Methods
[0878] At Day 0, C57BL/6J female mice were injected in the right
rear flank with TC1 tumor cells (50 k cells/mouse) at Day 0. On Day
7 (prime), splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. Splenocyte were loaded with pre-complexed 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25))+20 .mu.M
mouse serum albumin (MSA) via SQZ (60 psi; 4 .mu.m constriction)
and incubated with CpG 1826 (1 .mu.M in R10) for 4 hours. Female
C57BL/6J recipient mice (10/group) were injected retro-orbitally on
Day 7 with 100 .mu.L of either vehicle (PBS) or splenocytes
(0.05-1M cells/mouse). TC-1 tumor growth was measured beginning 1
week post-tumor implantation two times per week and compared to
tumor growth in untreated mice for 30 days.
Results
[0879] As shown in FIG. 9, Tumor growth, as measured by the formula
((length.times.width2)/2), was compared between mice from the
untreated group (no splenocytes) and groups treated with increasing
numbers of HPV E7-loaded splenocytes. It was found that the greater
the splenocyte dose, the better the tumor growth inhibition, with
the 1M dose leading to complete tumor regression on average. After
30 days, there were 5 remaining mice that were tumor-free in the 1M
treatment group, while the 0.25M group had 3 mice without tumors.
These data show that splenocytes loaded by SQZ can induce tumor
regression in a therapeutic model of HPV-associated cancer.
Example 10
[0880] In order to determine the effect of CpG maturation on B
cells within a mixed population, human PBMCs and murine splenocytes
were matured for various times with CpG, with the relative amount
of activation marker CD86 was measured in the B cell population by
flow cytometry and the levels of cytokines and chemokines were
quantified by a multiplex assay.
Methods
[0881] Murine splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. Human PBMCs and splenocytes were incubated in R10 with CpG
(2006 for human, 1826 for murine) for varying times (2-24 h) and
concentrations (1-10 .mu.M). After the CpG incubation, the cells
were washed with R10 and assessed for levels of CD86 by flow
cytometry, while supernatants were collected and the levels of
cytokines were analyzed using a multiplex (29-plex) human
cytokine/chemokine assay.
Results
[0882] FIG. 10A shows the following results: Human (Top)--The
higher 10 .mu.M dose of CpG showed that the levels of CD86 in the B
cell population of human PBMCs decreased over time after 2 hours.
The lower 1 .mu.M dose led to higher levels of CD86 than 10 .mu.M
for all time points, and it also exhibited a bimodal time course,
with levels decreasing after 2 h and beginning to return after 24
h. These data show that lower levels of CpG can lead to higher B
cell activation with all doses peaking at the earliest observed
time point.
[0883] Murine (Bottom)--For murine B cells, there was no
appreciable change in CD86 levels over time and at both
concentrations. These data show that murine B cells do not
upregulate CD86 in response to CpG maturation.
[0884] As shown in FIG. 10B, Cytokine/chemokine (FIG. 10B)--Results
from both human and mouse chemokine/cytokine profiles exhibit
similar trends, with many of the same proteins increasing in
response to CpG treatment (IFN-.gamma., IL-6, MIP-1B), although
IL-10 and IL-13 were increased only in murine splenocytes. These
results indicate that human PBMCs and murine splenocytes have
similar chemokine/cytokine responses to CpG, with IL-10 and IL-13
as notable exceptions.
Example 11
[0885] In order to determine the impact of SQZing on cell
composition and MHC-I levels, human PBMCs are subjected to cell
squeezing and the relative percentage of immune cells, as well as
surface expression of MHC-I, was assessed by flow cytometry.
Methods
[0886] Human PBMCs from HLA-A2+ donors were incubated with (Endo)
or loaded with fluorescently-labeled 3 kDa dextran (100 .mu.g/mL)
by SQZ (60 psi; 3.5-4 .mu.m width) at room temperature. The loaded
PBMCs were then analyzed for the relative composition of B cells, T
cells, NK cells and monocytes, as well as HLA-A2 MHC-I surface
expression via flow cytometry.
Results
[0887] As shown in FIG. 11, Loading human PBMCs using SQZ led to
changes less than 5% for B cells (CD20), T cells (CD3), NK cells
(CD56) and monocytes (CD14) [Top], coincident with a slight (12%)
decrease in MHC-I levels after SQZ. For both cell composition and
MHC-I levels, the 3.5 .mu.m constriction width led to marginally
higher alterations. Taken together, these data support that loading
PBMCs with SQZ does not appreciably alter the relative abundance of
the immune cell subsets and their MHC-I surface expression.
Example 12
[0888] In order to determine the delivery and impact of SQZing on
individual cell subsets, human PBMCs are subjected to cell
squeezing and the viability and delivery of a fluorescent compound
to the different immune cell populations was assessed by flow
cytometry.
Methods
[0889] Human PBMCs were incubated with (Endo) or loaded with
fluorescently-labeled 3 kDa dextran (100 .mu.g/mL) by SQZ (60 psi;
3.5-4 .mu.m width) at room temperature. The loaded PBMCs were then
analyzed for viability and delivery by flow cytometry.
Results
[0890] As shown in FIGS. 12A and B, Loading human PBMCs using SQZ
led to changes less than 10% for B cells (CD20), T cells (CD3), NK
cells (CD56) and monocytes (CD14) in viability [left]. The
percentage of cells delivered with dextran ranged between 60% (B
cells) to 90% (monocytes) for the 3.5 .mu.m width constriction,
with the 4 .mu.m width constriction led to .about.10-20% less cells
delivered across all cell types. There was up to a 35-fold increase
in the amount of dextran loaded per cell for T cells and monocytes
and .about.5-10-fold for B cells and NK cells for the 3.5 .mu.m
width. The 4 .mu.m width constriction generally decreased the
amount of delivery by .about.2-fold from the 3.5 .mu.m width.
Example 13
[0891] In order to determine the effect of delivery to individual
cell subsets on the overall functional response from a mixed
population, human PBMCs were loaded with a disease-relevant antigen
and a tagged dextran by SQZ and the ability to stimulate
antigen-specific responder cells will be measured and compared to
delivery of the tagged compound.
Methods
[0892] Human PBMCs from HLA-A02+ donors (10M cells/mL) were loaded
in the presence of 50 .mu.M E7 SLP (QLCTELQTYMLDLQPETTYCKQQLL (SEQ
ID NO: 23)) and fluorescently-labeled 3 kDa dextran (100 .mu.g/mL)
by SQZ (60 psi, room temperature) and the level of delivery to the
cell subsets between the differing constriction widths (3.5 and 4
.mu.m) were quantified by flow cytometry. PBMCs were then
co-cultured with E711-20-specific CD8+ responder cells in a ratio
of 2:1 stimulator:responder cells and cultured in the presence of
IL-2 (10 U/mL) and compared to untreated control or 2:1
stimulator:responder cells incubated with the minimal epitope
(PP--0.1 .mu.M--YMLDLQPETT (SEQ ID NO: 3)) overnight. After 24 h,
supernatant is harvested from each condition and the level of
IFN-.gamma. production was assessed by IFN-.gamma. ELISA.
Results
[0893] As shown in FIG. 13, loading human PBMCs using SQZ led to up
to a .about.4-fold increase (3.5 .mu.m) in the level of IFN-.gamma.
production assessed by ELISA (top). Delivery via the 4 .mu.m
constriction exhibited approximately half the level of
antigen-specific response to E7-specific responder T cells (middle
and right--re-analysis of FIG. 11 data from same samples). This
functional effect correlated with the higher delivery of E7 SLP by
the 3.5 .mu.m condition. These findings show that enhanced delivery
can lead to increases in the cell antigen-presentation
functionality of human PBMCs.
Example 14
[0894] In order to determine the impact of various delivery
parameters on the ability of immune cells to activate an
antigen-specific response, human PBMCs were loaded with a
disease-relevant antigen by SQZ and the ability to stimulate
antigen-specific responder cells will be measured and compared
across different SQZ conditions.
Methods
[0895] Human PBMCs from HLA-A02+ donors (10M cells/mL) were loaded
in the presence of 50 .mu.M pp65 SLP
(PPWQAGILARNLVPMVATVQGQNLKYQEFFWDAND (SEQ ID NO: 51)) by SQZ and
the pressure (45, 60 psi), temperature (room temperature ice) and
constriction widths (3.5-4.5 .mu.m) were altered. PBMCs were then
co-cultured with pp65-specific CD8+ responder cells in a ratio of
2:1 stimulator:responder cells, cultured in the presence of IL-2
(10 U/mL) and compared to untreated control or 2:1
stimulator:responder cells incubated with the minimal epitope
(PP--0.1 .mu.M--NLVPMVATV (SEQ ID NO: 55)) overnight. After 24 h,
supernatant is harvested from each condition and the level of
IFN-.gamma. production was assessed by IFN-.gamma. ELISA.
Results
[0896] As shown in FIG. 14, loading human PBMCs with pp65 SLP using
SQZ led to a .about.6-9-fold increase in the level of IFN-.gamma.
production assessed by ELISA. The narrower the constriction width
(3.5 .mu.m) and higher the pressure (60 psi) led to the higher
responses, with progressively wider chips leading to loss of
functionality, and this phenomenon is conserved between the room
temperature (top) and ice (bottom) conditions. Taken together,
there may be a slight benefit to ice during SQZ but all conditions
led to a significant increase in IFN-.gamma. production.
Example 15
[0897] In order to determine the impact of CpG maturation on the
ability of human immune cells to activate an antigen-specific
response and to compare this response to loaded T cell APCs, human
PBMCs or isolated T cells were loaded with a disease-relevant
antigen by SQZ and the ability to stimulate antigen-specific
responder cells was measured and compared to no CpG maturation.
Methods
[0898] Human HLA-A02+ PBMCs or T cells isolated from PBMCs of
HLA-A02+ donors (10M cells/mL) were loaded in the presence of 50
.mu.M pp65 SLP (PPWQAGILARNLVPMVATVQGQNLKYQEFFWDAND (SEQ ID NO:
51)) by SQZ (45 psi; 3 .mu.m constriction for PBMCs, 4.5 .mu.m used
for T cells). PBMCs were then co-cultured with pp65-specific CD8+
responder cells in a ratio of 2:1 stimulator:responder, cultured in
the presence of IL-2 (10 U/mL)+/-CpG 2006 (1 .mu.M) and compared to
untreated control or 2:1 stimulator:responder cells incubated with
the minimal epitope (PP--0.1 .mu.M--NLVPMVATV (SEQ ID NO: 55))
overnight. After 24 h, supernatant is harvested from each condition
and the level of IFN-.gamma. production was assessed by IFN-.gamma.
ELISA.
Results
[0899] As shown in FIG. 15, loading human PBMCs with pp65 SLP using
SQZ led to greater IFN-.gamma. production than cells that were
incubated with the SLP (Endo) both with and without CpG.
Additionally, there was a 30% increase in the response between SQZ
loaded conditions co-cultured with CpG relative to without
(P<0.05; top). T cells do not respond to CpG maturation, so the
T cell condition was compared directly to the PBMC co-cultured with
CpG conditions, and it was found that there was nearly double the
response in the PBMC condition (P<0.001; bottom). Taken
together, these data show that CpG co-culture enhances the
antigen-specific response of human PBMCs and that PBMCs with CpG
are nearly twice as potent at eliciting this response when compared
with loaded T cells.
Example 16
[0900] In order to examine the effect of adjuvant and antigen
concentration on the activation of an antigen-specific response in
the human context, human PBMCs were loaded with different
concentrations of a disease-relevant antigen by SQZ and the ability
to stimulate antigen-specific responder cells was measured and
compared among different adjuvants.
Methods
[0901] Human PBMCs from HLA-A02+ donors (10M cells/mL) were loaded
in the presence of different concentrations of pp65 SLP
(PPWQAGILARNLVPMVATVQGQNLKYQEFFWDAND (SEQ ID NO: 51); 1, 10 and 50
.mu.M) by SQZ (60 psi; 3.5 .mu.m constriction). PBMCs were then
co-cultured with pp65-specific CD8+ responder cells in a ratio of
2:1 stimulator:responder, cultured in the presence of IL-2 (10
U/mL)+/-CpG 2006 (1 .mu.M) or R837 (1 .mu.g/mL; imiquimod) and
compared to untreated control or 2:1 stimulator:responder cells
incubated with the minimal epitope (PP--0.1 .mu.M--NLVPMVATV (SEQ
ID NO: 55)) overnight. After 24 h, supernatant is harvested from
each condition and the level of IFN-.gamma. production was assessed
by IFN-.gamma. ELISA.
Results
[0902] As shown in FIG. 16, while there were no significant
differences between the function response of human PBMCs loaded
with 1 .mu.M pp65 SLP by SQZ with and without adjuvants, higher
concentrations did show a significant benefit to co-culture with
adjuvant. The 10 .mu.M pp65 SLP condition showed a slight but not
significant increase in the response of PBMCs co-cultured with CpG
relative to without, but there was a significant increase
(P<0.0001) when PBMCs co-cultured with R837 compared to no
adjuvant. This effect was even more pronounced when using 50 .mu.M
of SLP, where CpG and R837 led to significant enhancement of the
antigen-specific response (P<0.0001). In all cases, there was
increasing benefit to co-culturing with either adjuvant, although
R837 consistently led to the highest response, and this effect was
potentiated by higher concentrations of pp65 SLP. Taken together,
these data show that the use of an adjuvant during co-culture
enhances the antigen-specific response of human PBMCs and that this
effect is dependent on the concentration of antigen used.
Example 17
[0903] In order to examine the effect of adjuvant composition and
maturation duration on the activation of an antigen-specific
response in the human context, human PBMCs were loaded with a
disease-relevant antigen by SQZ, matured with different adjuvants
for different incubation times and the ability to stimulate
antigen-specific responder cells was measured and compared among
different adjuvants.
Methods
[0904] Human PBMCs from HLA-A02+ donors (10M cells/mL) were loaded
in the presence of 50 .mu.M pp65 SLP
(PPWQAGILARNLVPMVATVQGQNLKYQEFFWDAND (SEQ ID NO: 51)) by SQZ (60
psi; 3.5 .mu.m constriction). PBMCs were then matured with CpG 2006
(1 .mu.M), R837 (1 .mu.g/mL; imiquimod) or R848 (1 .mu.g/mL;
resiquimod) for either 3 or 24 h, followed by being co-cultured
with pp65-specific CD8+ responder cells in a ratio of 2:1
stimulator:responder, in the presence of IL-2 (10 U/mL) and
compared to untreated control or 2:1 stimulator:responder cells
incubated with the minimal epitope (PP--0.1 .mu.M--NLVPMVATV (SEQ
ID NO: 55)) overnight. After 24 h, supernatant is harvested from
each condition and the level of IFN-.gamma. production was assessed
by IFN-.gamma. ELISA.
Results
[0905] As shown in FIG. 17, There were no significant differences
between the function response of human PBMCs loaded with pp65 SLP
by SQZ with and without adjuvants, although the groups treated with
R848 for either 3 or 24 h did afford the highest overall response.
These data show that the use of an adjuvant to mature PBMCs
post-SQZ may enhance the antigen-specific response but this effect
was not found to lead to a significant increase in responses for
all adjuvants and time points tested.
Example 18
[0906] In order to quantify the impact of pre-SQZ or post-SQZ
maturation on antigen-specific response, murine splenocytes were
loaded with a model antigen, matured with CpG, and injected into
recipient mice once (Prime) or twice (Prime-Boost), with the
relative percentage of inflammatory cytokine IFN-.gamma.+CD8+ T
cells measured by flow cytometry.
Methods
[0907] On Day -1 (matur.fwdarw.SQZ) or Day 0 (SQZ.fwdarw.mater),
splenocytes were obtained from spleens of female C57BL/6J donor
mice, and either matured with CpG 1826 (1 .mu.M in R10) for 4 h on
Day -1 (matur.fwdarw.SQZ), then loaded with Ova protein (400
.mu.g/mL) by SQZ (30, 60, 90 psi; 4 .mu.m constriction) on Day 0
(SQZ.fwdarw.mater), or loaded with Ova by SQZ on Day 0 followed by
4 h incubation with CpG 1826. Female C57BL/6J recipient mice
(5/group) were injected retro-orbitally on Day 0 with 100 .mu.L of
splenocytes (1.times.10.sup.6 cells/mouse). On Day 7, spleens were
harvested, restimulated with SIINFEKL (SEQ ID NO: 54) (1 .mu.g/mL)
and the percentage of IFN-.gamma.+CD8+ T cells was determined by
intracellular cytokine staining (ICS).
Results
[0908] As shown in FIG. 18, While increasing pressure used to load
Ova into the splenocytes led to a significant increase in the
percentage of IFN-.gamma.+CD8+ T cells (P<0.05), there was no
significant change between splenocytes that were matured, then SQZ
loaded or were SQZ loaded and then matured. These data show that
there is no significant difference in the order of loading/maturing
splenocytes on the ability to elicit an antigen-specific response
in vivo.
Example 19
[0909] In order to determine if there is a benefit to co-treating
animals with antigen-loaded splenocytes in combination with
platinum-based chemotherapy, tumor growth inhibition of
antigen-associated tumor cells was measured in an in vivo
therapeutic model, with multiple splenocyte+/-chemotherapy
treatment regimes compared, with the survival of mice in each group
plotted against time.
Methods
[0910] At Day 0, C57BL/6J female mice were injected in the right
rear flank with TC1 tumor cells (50 k cells/mouse; 10 mice/group)
at Day 0. On Days 5 and 7, mice were either injected with vehicle
or cisplatin (5 mg/kg) according to the groups. On Day 9, some
animals also received a prime of splenocytes obtained from spleens
of female C57BL/6J donor mice that were loaded with pre-complexed
20 .mu.M E7 SLP (GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO:
25))+20 .mu.M mouse serum albumin (MSA) via SQZ (60 psi; 4 .mu.m
constriction) and incubated with CpG 1826 (1 .mu.M in R10) for
.about.16 h. On Days 27 and 29, untreated mice or mice treated with
splenocytes only on Day 9 were given 1-2 doses of cisplatin
according to the groups outlined. The survival of each group of TC1
tumor-bearing mice was assessed and plotted over 120 days.
Results
[0911] As shown in FIG. 19, tumor growth, as measured by the
formula ((length.times.width2)/2), was compared between mice that
were untreated, treated with E7-loaded splenocytes alone, 1-2 doses
of cisplatin alone or various combinations and the Kaplan-Meier
survival curves were plotted. The four treatment groups that
included splenocytes exhibited a large survival advantage over not
just untreated animals, but those receiving cisplatin alone, with a
median survival time beyond 50 days (and in the case of
splenocyte+cisplatin treatment, the median survival time had still
not been reached after 120 days). Of particular note, the
splenocyte alone treated mice had a .about.40 days survival
advantage over the untreated and cisplatin alone groups, but the
splenocyte+cisplatin groups had a larger survival advantage even
over the splenocyte alone group. These data show that splenocytes
loaded by SQZ can induce a survival advantage in a therapeutic
model of HPV-associated cancer, and that the addition of cisplatin
chemotherapy further potentiated the survival advantage of the
splenocyte vaccine.
Example 20
[0912] In order to determine the delivery and impact of different
SQZing parameters on individual cell subsets using on a
clinical-scale, human PBMCs are subjected to cell squeezing at
different temperatures and pressures and the viability and delivery
of a fluorescent compound to the different immune cell populations
was assessed by flow cytometry.
Methods
[0913] Human PBMCs were loaded with fluorescently-labeled 3 kDa
dextran (100 .mu.g/mL) by SQZ (50-70 psi; 4.5 .mu.m width) at room
temperature and on ice. The loaded PBMCs were then analyzed for
viability and delivery by flow cytometry.
Results
[0914] As shown in FIGS. 20A and B, loading human PBMCs using SQZ
on a clinical scale still allowed for successful delivery of up to
80% of cells (monocytes on ice--FIG. 20A), with cells SQZ'd on ice
leading to higher percentages (30-50% increase) of delivered cells
across all cell subsets. The higher pressure (70 psi) afforded the
highest percentage of delivered cells relative to 50 psi, but in
all cases tested the viability was above 88% for the bulk PBMC
population. Taken together, these data show that SQZ can be used to
deliver to multiple cell types in a mixed population, and that
SQZing on ice at slightly higher pressures led to the best overall
delivery.
Example 21
[0915] In order to determine the impact the
maturation+/-co-injection of adjuvant on the ability of
antigen-loaded splenocytes to lead to tumor growth inhibition in a
therapeutic setting, splenocytes were either matured with,
co-injected with or matured and co-injected with adjuvant and
tested in the HPV E7-expressing TC1 tumor model, with the area of
the tumors and survival plotted against time.
Methods
[0916] At Day 0, C57BL/6J female mice were injected in the right
rear flank with TC1 tumor cells (50 k cells/mouse). On Day 10
(prime), splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation to better
mimic human PBMCs, leading to a splenocyte composition more
representative of human PBMCs (i.e., crafted splenocytes).
Splenocyte were loaded with pre-complexed 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25))+20 .mu.M
mouse serum albumin (MSA) via SQZ (60 psi; 4 .mu.m constriction,
room temperature) and incubated with CpG 1826 (1 .mu.M in R10) for
4 hours. Female C57BL/6J recipient mice (10/group) were injected
retro-orbitally on Day 10 with 100 .mu.L of either vehicle (PBS),
splenocytes (1M cells/mouse) or splenocytes+CpG (1 .mu.g/mouse).
TC-1 tumor growth was measured beginning 1 week post-tumor
implantation two times per week and compared to tumor growth in
untreated mice for 32 days.
Results
[0917] Tumor growth, as measured by the formula
((length.times.width.sup.2)/2), was compared between mice from the
untreated group (no splenocytes) and groups treated with adjuvant
alone (CpG), splenocytes or splenocytes+co-injected adjuvant. As
shown in FIGS. 21A and B, while there was no observable difference
between the tumor growth of untreated animals and those treated
with CpG alone (median survival of 28 and 32 days, respectively),
there was a slight inhibition in the rate of tumor growth for
un-matured splenocytes loaded with E7. However, the groups that
received either matured, loaded splenocytes+/-co-injection of CpG
led to tumor regression, with tumors not reaching their initial
maximum over the course of the study. Additionally, none of the
splenocyte-treated groups reached the median survival point by day
32. These data show that splenocytes loaded by SQZ and matured with
adjuvant (with or without adjuvant co-injection) can induce tumor
regression in a therapeutic model of HPV-associated cancer.
Example 22
[0918] In order to determine if antigen-loaded splenocytes
co-injected with different adjuvants can elicit an antigen-specific
response, splenocytes were loaded with a model antigen and either
matured with CpG, co-injected with CpG or matured with CpG and
co-injected with either CpG or IFN-.alpha., with mice receiving
prime and boost, and the relative percentage of inflammatory
cytokine IFN-.gamma.+CD8+ T cells was measured by flow
cytometry.
Methods
[0919] At Day 0, splenocytes were obtained from spleens of female
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs. These mixed splenocytes were then loaded with Ova protein
(400 .mu.g/mL) by SQZ (60 psi; 4 .mu.m constriction) and incubated
in either media (R10) alone or media with CpG 1826 (1 .mu.M) for 4
h. Female C57BL/6J recipient mice (5/group) were injected
retro-orbitally on Day 1 with 100 .mu.L of either vehicle (PBS),
splenocytes (1.times.10.sup.6 cells/mouse) matured with CpG,
splenocytes co-injected with 1 .mu.g CpG1826 or splenocytes matured
with CpG that are co-injected with either CpG or 10000 U
IFN-.alpha.. On Day 7, recipient mice were boosted in an identical
fashion to the prime on Day 0. On Day 14, spleens were harvested,
restimulated with SIINFEKL (SEQ ID NO: 54) (1 .mu.g/mL) and the
percentage of IFN-.gamma.+CD8+ T cells was determined by
intracellular cytokine staining (ICS).
Results
[0920] As shown in FIG. 22, the percentage of IFN-.gamma.+CD8+ T
cells for mice treated with Ova-loaded splenocytes was highest when
splenocytes were matured with CpG for 4 h with or without
co-injection of CpG, with a significant increase when compared to
untreated (P<0.0001), unmatured splenocytes (P<0.005), as
well as unmatured splenocytes+co-injected CpG (P<0.05). While
there was a slight increase in the percentage of IFN-.gamma.+CD8+ T
cells, there was no significant benefit to unmatured splenocytes
co-injected with CpG or matured splenocytes co-injecting with
IFN-.alpha. relative to untreated. These data show that there is a
requirement for CpG maturation for a significant increase in
inflammatory cytokine production in antigen-specific CD8+ T cells,
and that co-injection of CpG was slightly better than co-injection
of IFN-.alpha..
Example 23
[0921] In order to assess the upregulation of B cell maturation
markers in PBMCs after maturation with CpG 1826 following SQZ
processing, the upregulation of B cell maturation markers was
measured by flow cytometry after SQZ-processed murine splenocytes
were incubated with CpG1826.
Methods
[0922] Splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation, leading to a
splenocyte composition more representative of human PBMCs (i.e.,
crafted splenocytes). The crafted murine splenocytes were then
SQZ-processed without payload and the levels of CD86 and H-2Kb were
measured by flow cytometry.
Results
[0923] As shown by the mean fluorescence intensity (MFI) in FIGS.
23A-G, four independent experiments demonstrated increased CD86 and
murine MHC-I (H-2Kb) expression on B220+ cells (B cells) within the
crafted murine splenocytes following CpG 1826 maturation. The
increase in CD86 and H-2Kb expression for B220+ cell subsets (B
cells) subsequent to maturation with CpG1826 was similar for both
SQZ-processed crafted murine splenocytes (gray bars) and
unprocessed crafted murine splenocytes (black bars). These data
indicate that SQZ-processing did not alter the effects of CpG on
the maturation of B220+ cells (B cells) within crafted murine
splenocytes.
Example 24
[0924] In order to determine if antigen-loaded PBMCs co-injected
with adjuvants will elicit systemic effects in serum cytokine
levels, murine splenocytes were loaded with an HPV antigen and
matured with CpG, and introduced into mice with or without CpG
co-injection, and the circulating cytokines in mice were measured
by multiplexed cytokine assays.
Methods
[0925] Splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation, leading to a
splenocyte composition more representative of human PBMCs (i.e.,
crafted splenocytes). The crafted murine splenocytes were
SQZ-loaded with 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25)), followed by
incubation with CpG 1826 (1 .mu.M in R10) for 4 hours at 37.degree.
C. Female C57BL/6J recipient mice were either injected with the
crafted murine splenocytes SQZ-loaded with E7 SLP
(M-SQZ-Spleno-HPV), or injected with 1 .mu.g of CpG 1826 IV (No
cells), or with a combination of both (M-SQZ-Spleno-HPV+1 .mu.g
co-injected CpG), and the levels of circulating cytokines were
measured from blood via a murine multiplex (25-plex)
cytokine/chemokine assay.
Results
[0926] As shown in FIGS. 24A-24D, the ranges of serum cytokine
concentrations in mice immunized with SQZ-loaded crafted murine
splenocytes (FIG. 24C, 24D) were comparable to the ranges of serum
cytokine concentrations measured in the no treatment control mice
(FIG. 24A) and in mice injected with 1 .mu.g CpG 1826 IV (No cells)
(FIG. 24B) at all timepoints. These results indicate that
immunization with M-SQZ-Spleno-HPV with or without CpG 1826
co-injection did not lead to changes in either the production or
serum concentrations of pro-inflammatory cytokines relative to no
treatment or CpG (No Cells) controls. The presence of serum
cytokines in all conditions further demonstrated immunization with
M-SQZ-Spleno-HPV had no systemic effects in serum cytokine
production or secretion.
Example 25
[0927] To investigate the impact of SQZ-mediated processing of
PBMCs on circulation kinetics upon adoptive transfer, murine
splenocytes SQZ-loaded with E7 SLP (M-SQZ-Spleno-HPV) and
unprocessed murine splenocytes were respectively administered to
mice via intravenous injection and the number of circulating donor
murine splenocytes in blood over time was determined by flow
cytometry.
Methods
[0928] Splenocytes were obtained from spleens of female C57BL/6J
(CD45.1) donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs (i.e., crafted splenocytes). The crafted murine splenocytes
were then SQZ-loaded with 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25)), followed by
incubation with CpG 1826 (1 .mu.M in R10) for 4 hours at 37.degree.
C., and the loaded crafted murine splenocytes were injected
retro-orbitally into female CD45.2 B6.SJL-Ptprca Pepcb/BoyJ
recipient mice (5-7 mice/group), with blood (100 uL) collected from
recipient mice over the course of two weeks post-administration at
the following timepoints: 30 minutes post-administration, Day 1,
Day 3, Day 7, and Day 15. The number of circulating crafted murine
splenocytes over time were assessed by flow cytometry.
Results
[0929] As shown in FIG. 25, upon adoptive transfer,
M-SQZ-Spleno-HPV and the unprocessed crafted murine splenocytes
exhibited similar persistence in the host blood over the course of
two weeks post-immunization (cumulative data displayed from 4
independent experiments). By one-way ANOVA and Tukey post-hoc
tests, there is no statistical difference between the two groups at
any timepoint. These studies demonstrate that the circulation
kinetics of M-SQZ-Spleno-HPV is not statistically different from
unprocessed crafted murine splenocytes.
Example 26
[0930] The objective of this study was to quantify E7-specific CD8+
T cells in the tumor microenvironment of TC-1 tumors 12 days post
immunization with M-SQZ-Spleno-HPV, compared to control cells, and
to correlate the E7-specific CD8+ T cells with tumor clearance in a
tumor growth model.
Methods
[0931] On Day 0, TC-1 cells (50 k/mouse in 100 .mu.L of PBS) were
injected subcutaneously in the right rear flank of female C57BL/6J
mice (7/group). On Day 16, splenocytes were obtained from spleens
of female C57BL/6J donor mice, and combined with splenocytes that
have had their B cells depleted by negative immunomagnetic
separation, leading to a splenocyte composition more representative
of human PBMCs (i.e., crafted splenocytes). The crafted murine
splenocytes were SQZ-loaded with 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25)), incubated
with CpG 1826 (1 .mu.M in R10) for 4 hours at 37.degree. C. and the
loaded crafted murine splenocytes were injected retro-orbitally
into the tumor bearing mice. On Day 28 (12 days post-immunization),
tumors were excised and from which a single-cell suspension was
generated. The single-cell suspension was assessed for
tumor-infiltrating lymphocytes (TILs) by flow cytometry.
Results
[0932] As seen in FIG. 26A, mice immunized with M-SQZ-Spleno-HPV
had a 44.7-fold increase in the percentage of CD8+ T cells in the
tumor compared to control mice (no treatment) 12 days after
immunization (i.e. 28th day post tumor implant). In
M-SQZ-Spleno-HPV treated animals, the majority of these CD8+ T
cells were specific for the E7 antigen as determined by tetramer
staining (87.2.+-.6.0% of the CD8.alpha.+ population) (FIG. 26B).
The percentage of cells positive for E-7 tetramer staining in the
tumor was also quantified (FIG. 26C). This demonstrates that
immunization with M-SQZ-Spleno-HPV significantly increased the
presence of E7-specific CD8+ T cells in the tumor microenvironment
12 days post immunization in comparison to the no treatment group
(765-fold increase). As shown in FIGS. 26D and 26E, this increase
in E7 specific CD8+ T cells was also observed when normalized to
tumor mass. These data demonstrate that immunization with
M-SQZ-Spleno-HPV in the TC-1 mouse tumor model led to a significant
increase in E7-specific CD8+ T cells infiltrating the tumor. The
increase in E7-specific CD8+ T cells (FIGS. 26A-E) coupled with the
decrease in tumor volume (FIG. 26F) supported that M-SQZ-Spleno-HPV
reduced tumor burden by expanding E7-specific effector CD8+ T
cells.
Example 27
[0933] In order to demonstrate whether the murine splenocytes
processed with a model antigen (Ova) stimulates OT-I CD8+ T cell
proliferation in vivo through direct presentation of SIINFEKL (SEQ
ID NO: 54) (the CD8-restricted epitope of ovalbumin), MHC-I -/-
mice were used as recipients to decouple antigen hand-off to
professional APCs in recipient mice to allow examination of direct
presentation by murine splenocytes SQZ-processed with OVA.
Methods
[0934] On Day 0, OT-I T cells were isolated from the splenocytes of
female OT-I mice by immunomagnetic separation. Isolated OT-I T
cells were stained with CFSE prior to injection into female
recipient mice (WT or MHC-I -/-) retro-orbitally (2.5*10.sup.6
cells/mouse). Next, splenocytes were obtained from spleens of
female CD45.1 C57BL/6J donor mice, and combined with splenocytes
that have had their B cells depleted by negative immunomagnetic
separation, leading to a splenocyte composition more representative
of human PBMCs (i.e., crafted splenocytes). The crafted murine
splenocytes were SQZ-loaded with Ova (400 .mu.g/mL), incubated with
CpG 1826 (1 .mu.M in R10) for 4 hours at 37.degree. C. and the
loaded crafted murine splenocytes were injected retro-orbitally
into recipient mice (5*10.sup.6 cells/mouse). On Day 3, lymph nodes
and spleens were excised from the recipient mice and assessed for
proliferation using flow cytometry and CFSE staining.
Results
[0935] As shown in FIGS. 27A-D, both C57BL/6J (WT) and MHC-I-/-
mice that received the crafted murine splenocytes SQZ-loaded with
ovalbumin exhibited robust OT-I CD8+ T cell proliferation, with a
proliferation index ranging from 4-6 in both spleen and lymph node.
WT and MHC-I-/- mice that received the crafted murine splenocytes
incubated with ovalbumin (incubation control, without SQZ
processing) had little to no OT-I CD8+ T cell proliferation,
demonstrating that the SQZ process is required to introduce antigen
into crafted murine splenocytes for direct presentation to CD8+ T
cells in vivo. Mice that received OT-I T cells only (control for
unstimulated adoptively transferred OT-I CD8+ T cells) induced no
proliferation of CD8+ T cell. These results demonstrate that MHC-I
presentation is restricted to the transferred crafted murine
splenocytes processed with ovalbumin, ruling out the possibility
for antigen handoff to recipient antigen presenting cells (APCs)
playing a role in the subsequent CD8+ T cell response.
Proliferation of OT-I CD8+ T cells in MHC-I-/- recipients
demonstrates that the SQZ-processed crafted murine splenocytes
directly presents antigen.
Example 28
[0936] This study examined the antigen presentation ability of four
cell types within crafted murine splenocytes (B cells, T cells, NK
cells, and monocytes) by assessing activation of antigen-specific T
cells upon co-culture.
Methods
[0937] On Day 0, splenocytes were obtained from spleens of female
CD45.1 C57BL/6J donor mice, and combined with splenocytes that have
had their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs (i.e., crafted splenocytes). Crafted murine splenocytes were
then SQZ-processed with or without Ova (400 .mu.g/mL), and
incubated with CpG 1826 (1 .mu.M in R10) for 4 hours at 37.degree.
C. After 4 hours, aliquots of the SQZ-loaded crafted murine
splenocytes (5*10.sup.6 cells/mouse) were subjected to various
immunomagnetic separations to obtain purified subsets of monocytes,
B cells, T cells and NK cells. Next, OT-I T cells were isolated
from the splenocytes of female OT-I mice by immunomagnetic
separation. OT-I (1*10.sup.5 cells/well) were co-cultured with
either the crafted murine splenocytes or the respective individual
cell subsets (SQZ-processed with or without OVA) and incubated for
24 h at 37.degree. C. For positive control (Peptide Spike),
SIINFEKL (SEQ ID NO: 54) peptide (OVA257-264-1 .mu.g/mL) was added
into the suspension with OT-I and unprocessed crafted murine
splenocytes and kept for the entire duration of the co-culture. The
T cell activation marker CD69 was then assessed by flow
cytometry.
Results
[0938] Cell surface CD69 expression was used as a readout of OT-I
CD8+ T cell activation, because the surface expression of this
marker increases following engagement of the T cell receptor by
peptide antigen presented in the context of MHC-I. As shown in
FIGS. 28A-28E, all four major cell types within the crafted murine
splenocytes (B cells, T cells, NK cells, and monocytes) were
capable of directly presenting antigen to OT-I CD8+ T cells. These
data indicate that each of these cell types can function as antigen
presenting cells. All cell types SQZ-processed with Ova and matured
with CpG 1826 presented antigen to the OT-I CD8+ T cells and
induced activation of OT-I T cells at levels comparable to the
positive control (Peptide Spike).
Example 29
[0939] This study determined if fluorescently labeled HPV E6 or E7
SLPs, or the combination thereof, can be delivered intracellularly
through SQZ processing and whether any delivered SLPs are localized
in the cytosol of human PBMCs.
Methods
[0940] Human PBMCs from 3 different HLA-A*02+ donors were
SQZ-loaded with 50 .mu.M FAM-labeled E6, E7 or the combination of
E6+E7 on ice and the cells were co-stained with an AF647-conjugated
anti-CD45 antibody (plasma membrane marker) stain and Hoechst 33342
staining (nuclear staining). Human PBMCs SQZ processed with RPMI
only served as the negative control. The localization of the
peptides was determined by confocal imaging. Specifically,
localization of any FAM-E6 and/or FAM-E7 SLP was visualized on a
scanning disk confocal microscope. Z-stack analysis was performed
on the cells to determine the precise localization of FAM-E6 and/or
FAM-E7 SLP. Line scan traces were performed on confocal slices from
the middle of a Z-stack for each sample (i.e. the image of such a
slice depicted the middle of the cell) and these slices were
analyzed to confirm any intracellular delivery of SLPs following
SQZ processing. For the line scan traces, cells with a clear FAM
signal were selected for analysis, where the line scan was traced
through the center of the cell (white circle), along the white
lines displayed on the fluorescent images (FIGS. 29A-29F, top
panels).
Results
[0941] Following SQZ processing, the FAM fluorescent signal was
observed to be encircled by the plasma membrane within the optical
slices examined (FIGS. 29B, 29D, 29F, top three panels), whereas no
FAM signals were detected in negative controls (FIGS. 29A, 29C,
29E, top three panels). Most SQZ-loaded cells displayed visible
intracellular SLP of varying intensities in the widefield images;
however, dimmer signals could be difficult to visualize due to the
dynamic range of the microscope. Line scan traces showed that the
majority of the FAM fluorescent signal was detected within the
confines of the plasma membrane signal (FIGS. 29B, 29D, 29F, bottom
panels) when comparing to traces of negative controls which only
showed the plasma membrane signal (FIGS. 29A, 29C, 29E, bottom
panels), indicating that the FAM-E6 and FAM-E7 SLPs were
intracellular following SQZ processing. This indicates that the SQZ
process loaded the respective SLPs into the cytosol, though some
signal was detected to colocalize with the nucleus (not shown). Of
note, FAM molecules are typically cleaved from immunogenic epitopes
during the proteasomal processing of SLPs for presentation on
MHC-I. Thus, in live systems, the FAM signals from FAM-labeled SLPs
will be uncoupled from SLP as it is processed and presented on
MHC-I. However, in these experiments human PBMCs were fixed and
then stained immediately after SQZ processing to minimize such
processing of the FAM-labelled SLPs. This confocal imaging study
confirms the intracellular delivery of fluorescently labeled E6 and
E7 SLPs (FAM-E6 and FAM-E7) into human PBMCs by the SQZ
process.
Example 30
[0942] In order to determine if a disease-relevant antigen loaded
into murine B cells can lead to proliferation of antigen-specific T
cells, gp100 was loaded into B cells by SQZ-processing, and the
proliferation of gp100-specific T cells (pmel CD8.sup.+ T cell) was
analyzed by flow cytometry. Methods
[0943] Murine B cells were left untreated (NC), incubated at room
temperature with gp100 synthetic long peptide (SLP) (Incub. ctrl),
SQZ-processed in the presence of the gp100 SLP (Squeeze), or pulsed
with short peptide for 1 h at 37.degree. C. (PP). B cells
(5.times.10.sup.6 cells/mouse) were co-injected with 3 .mu.g LPS to
immunize mice that had also received 2.5.times.10.sup.6 CFSE
labeled pmel CD8.sup.+ T cells. To measure for proliferation, CFSE
dilution in the pmel CD8.sup.+ T cells was assessed 3 days after
immunization (n=5 mice per group).
Results
[0944] Murine B cells loaded with gp100 by SQZ-processing led to
significant increases in gp100-specific T cell proliferation, as
shown by the increased CFSE dilution (FIG. 30 left panel) and
subsequent quantification of proliferation index (FIG. 30 right
panel). SQZ-loaded B cells had a near 5-fold increase in
proliferation relative to untreated controls, peptide pulse control
or endocytosis control (FIG. 30 right panel). These data show that
SQZ-loading of antigen into B cells leads to significantly more
efficient stimulation of antigen-specific T cell proliferation than
incubating B cells with SLP or the minimal epitope.
Example 31
[0945] In order to determine if splenocytes SQZ-loaded with a
synthetic long peptide (SLP) can prime a protective immune
response, crafted splenocytes were SQZ-processed in the presence of
E7 synthetic long peptide (E7 SLP) and subsequently administered to
mice, at both 14 days and 7 days prior to implantation of HPV16
E6/E7 expressing tumor cell line TC-1.
Methods
[0946] Splenocytes were obtained from spleens of female CD45.1
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs (i.e., crafted splenocytes). The crafted splenocytes were
then SQZ-processed in the presence of a SLP containing a CD8
epitope for HPV16 E7. Mice were primed on day -14 and boosted on
day -7 with crafted splenocytes SQZ-loaded with the E7 SLP. On day
0, mice were subcutaneously implanted on the right flank with the
HPV16 E6/E7-positive TC-1 tumor cell line. Tumor growth, as
measured by the formula ((length.times.width2)/2), was compared
between mice from the untreated groups (Cohort I and Cohort II) and
a group treated with HPV E7-loaded crafted splenocytes. All
immunized mice remained tumor free for 60 days and were
subsequently rechallenged with TC-1 tumor cells subcutaneously
implanted on the left flank, and compared to a different cohort of
untreated animals implanted with tumor cells on the left flank.
Results
[0947] As shown in FIG. 31, all mice treated with SQZ-loaded
crafted splenocytes (15/15) were protected from the primary tumor
challenge while untreated animals invariably developed tumors. Upon
rechallenge, 11/15 mice treated with SQZ-loaded crafted splenocytes
were fully protected from tumor growth which was consistent with
the formation of protective immunological memory.
Example 32
[0948] To examine the effect of boosting using varying doses of E7
SLP-loaded crafted splenocytes, squeezed with E7 synthetic long
peptide. Mice were administered with the indicated doses of
squeezed splenocytes SQZ-loaded with E7 SLP, either as a prime only
immunization on day 10 post implant, or as a prime/boost/boost
regimen administered on days 10, 17, and 24.
Methods
[0949] At Day 0, C57BL/6J female mice were injected in the right
rear flank with TC1 tumor cells (50 k cells/mouse). On Day 10
(prime), splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation to better
mimic human PBMCs, leading to a splenocyte composition more
representative of human PBMCs (i.e., crafted splenocytes). The
crafted plenocytes were loaded with pre-complexed 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25))+20 .mu.M
mouse serum albumin (MSA) via SQZ (60 psi; 4 .mu.m constriction,
room temperature) and incubated with CpG 1826 (1 .mu.M in R10) for
4 hours. Female C57BL/6J recipient mice (10/group) were injected
retro-orbitally on Day 10 with 100 .mu.L of either vehicle (PBS),
splenocytes (1M cells/mouse) or splenocytes (1M cells/mouse)+CpG (1
.mu.g/mouse).
[0950] To examine the effect of booster immunizations, a cohort of
the therapeutically immunized mice received additional doses of
SQZ-loaded splenocytes on days 17 and 24 (Prime/boost/boost). Tumor
growth, as measured by the formula ((length.times.width2)/2), was
compared between untreated mice and the indicated treatment groups
until Day 50.
Results
[0951] As shown in FIG. 32, a single dose of 0.1.times.10.sup.6
SQZ-loaded splenocytes administered on Day 10 had modest efficacy
in priming immune response; however, boosting with additional doses
of 0.1.times.10.sup.6 cells enhanced therapeutic efficacy
significantly. On the other hand, dosage(s) of 1.0.times.10.sup.6
cells delayed tumor growth when administered as either a single
priming dose prime or under a prime and boost regimen.
Example 33
[0952] In order to determine the impact the
maturation+/-co-injection of adjuvant on the ability of
antigen-loaded splenocytes to lead to tumor growth inhibition in a
therapeutic setting, splenocytes were either matured with,
co-injected with or matured and co-injected with adjuvant and
tested in the HPV E7-expressing TC1 tumor model, with the area of
the tumors and survival plotted against time.
Methods
[0953] At Day 0, C57BL/6J female mice were injected in the right
rear flank with TC1 tumor cells (50 k cells/mouse). On Day 10
(prime), splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation to better
mimic human PBMCs, leading to a splenocyte composition more
representative of human PBMCs (i.e., crafted splenocytes). Crafted
splenocytes were loaded with pre-complexed 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25))+20 .mu.M
mouse serum albumin (MSA) via SQZ (60 psi; 4 .mu.m constriction,
room temperature) and incubated with CpG 1826 (1 .mu.M in R10) for
4 hours. Female C57BL/6J recipient mice (10/group) were injected
retro-orbitally on Day 10 with 100 .mu.L of either vehicle (PBS),
splenocytes (1M cells/mouse) or splenocytes+CpG (1 .mu.g/mouse).
TC-1 tumor growth was measured beginning 1 week post-tumor
implantation two times per week and compared to tumor growth in
untreated mice for 60 days.
Results
[0954] Tumor growth, as measured by the formula
((length.times.width.sup.2)/2), was compared between mice from the
untreated group (no splenocytes) and groups treated with adjuvant
alone (CpG), splenocytes or splenocytes+co-injected adjuvant. As
shown in FIGS. 33A and B, while there was no observable difference
between the tumor growth of untreated animals and those treated
with CpG alone (median survival of 28 and 32 days, respectively),
there was a slight inhibition in the rate of tumor growth for
un-matured splenocytes loaded with E7 (median survival of 35 days).
However, the groups that received matured, loaded splenocytes,
either with or without co-injection of CpG, led to tumor
regression, with tumors not reaching their initial maximum over the
course of the study (median survival of 53 and 56 days,
respectively). These data show that splenocytes loaded by SQZ and
matured with adjuvant (with or without adjuvant co-injection) can
induce tumor regression in a therapeutic model of HPV-associated
cancer.
Example 34
[0955] To assess MHC-I presentation of SQZ-delivered antigens by
major cell subsets contained within mouse splenocytes, crafted
murine splenocytes were SQZ-loaded with OVA and subsequently the
presentation in subsets were analyzed via flow cytometry using
25-D1.16 antibody. The 25-D1.16 antibody specifically binds to
H-2Kb (MHC-I) on the surface of mouse cells only when it presents
the immunodominant CD8.sup.+ T cell epitope from ovalbumin
(SIINFEKL (SEQ ID NO: 54)).
Methods
[0956] Splenocytes were obtained from spleens of female CD45.1
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs (i.e., crafted splenocytes). The crafted splenocytes were
then SQZ-processed without cargo (SQZ only) or SQZ-processed in the
presence of ovalbumin protein (SQZ+OVA). At the indicated time
points, cells were stained with an antibody panel that included the
25-D1.16 antibody to assess SIINFEKL (SEQ ID NO: 54) presentation
on H-2Kb for each indicated cell type, and analyzed by flow
cytometry (T cells, B cells, NK cells, and monocytes).
Results
[0957] As shown in FIG. 34, for cells SQZ-processed in the presence
of OVA, antigen presentation was detectable at the 2- and 4-hour
time points for monocytes, T cells, B cells, and NK cells. This was
evident from the increased signal from the 25-D1.16 antibody
staining relative to cells that were not SQZ-processed with OVA
(SQZ only). At 2 and 4 hours after SQZ-processing, an increase in
fluorescence intensity of >40% was detectable for T cells, B
cells, and NK cells. For monocytes, the increase in fluorescence
intensity was >10% at 2 and 4 hours after SQZ-processing.
Example 35
[0958] In order to demonstrate whether murine splenocytes processed
with a tumor antigen (HPV16 E7) stimulates E7-specific T cell
proliferation, mice were immunized with crafted murine splenocytes
were SQZ-loaded with E7 SLP, and endogenous T cell response upon
antigen re-stimulation was measured by intracellular cytokine
staining.
Methods
[0959] Splenocytes were obtained from spleens of female CD45.1
C57BL/6J donor mice, and combined with splenocytes that have had
their B cells depleted by negative immunomagnetic separation,
leading to a splenocyte composition more representative of human
PBMCs (i.e., crafted splenocytes). The crafted murine splenocytes
were SQZ-loaded with E7 SLP (20 .mu.M), incubated with CpG 1826 (1
.mu.M in R10) for 4 hours at 37.degree. C. and the loaded crafted
murine splenocytes were injected retro-orbitally into C57BL/6J
recipient mice at 1 million, 0.25 million or 0.1 million cells per
mouse. Control mice were left untreated. 7 days after immunization,
spleens were harvested from the recipient mice and re-stimulated
with the E7 minimal epitope ex vivo. Intracellular cytokine
staining was performed to determine the percentage of endogenous
CD8 T-cells that produced interferon-.gamma. in response to E7
recognition.
Results
[0960] As shown in FIG. 35, mice that received 1 million crafted
murine splenocytes SQZ-loaded with E7 SLP exhibited robust
E7-specific CD8+ T cell proliferation in the spleen, as illustrated
by the significant induction of IFN-.gamma. secretion when
recipient mouse spleen was stimulated with the E7 minimal epitope
ex vivo. Mice that received 0.25 million crafted murine splenocytes
SQZ-loaded with E7 SLP also exhibited E7-specific CD8+ T cell
proliferation in the spleen, as illustrated by the observable
induction of IFN-.gamma. when recipient mouse spleen was stimulated
with the E7 minimal epitope ex vivo. In contrast, untreated animals
did not exhibit any observable E7-specific CD8+ T cell
proliferation, as illustrated by the lack of IFN-.gamma. secretion
when spleen was stimulated with the E7 minimal epitope ex vivo.
Example 36
[0961] The objective of this study was to quantify E7-specific CD8+
T cells in the tumor microenvironment of TC-1 tumors 12 days post
immunization with M-SQZ-Spleno-HPV, or with a peptide vaccine (E7
SLP+CpG), compared to control cells, and to correlate the
E7-specific CD8+ T cells with tumor clearance in a tumor growth
model.
Methods
[0962] On Day 0, TC-1 cells (50 k/mouse in 100 .mu.L of PBS) were
injected subcutaneously in the right rear flank of female C57BL/6J
mice (7/group). On Day 16, splenocytes were obtained from spleens
of female C57BL/6J donor mice, and combined with splenocytes that
have had their B cells depleted by negative immunomagnetic
separation, leading to a splenocyte composition more representative
of human PBMCs (i.e., crafted splenocytes). The crafted murine
splenocytes were SQZ-loaded with 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25)), incubated
with CpG 1826 (1 .mu.M in R10) for 4 hours at 37.degree. C. and the
loaded crafted murine splenocytes were injected IV
(retro-orbitally) into the tumor bearing mice. For mice receiving a
peptide vaccine, 150 .mu.g of E7 SLP and 50 .mu.g of CpG were
injected subcutaneous (per mouse) to the recipient mice on Day 16.
Control mice were left untreated.
[0963] On Day 28 (12 days post-immunization), tumors were excised
and from which a single-cell suspension was generated. The
single-cell suspension was assessed for tumor-infiltrating
lymphocytes (TILs) by flow cytometry.
Results
[0964] As seen in FIG. 36A, mice immunized with M-SQZ-Spleno-HPV
had a significant increase in CD45+ leukocytes among live cells
within the tumor microenvironment, compared to mice receiving
peptide vaccine or control mice at 12 days after immunization (i.e.
28th day post tumor implant). Of these leukocytes in the tumor,
mice immunized with M-SQZ-Spleno-HPV had a significantly higher
percentage of CD8+ T cells (>30%), compared to control mice
(<5%) and mice receiving peptide vaccine (<20%) (FIG. 36B).
Furthermore, in M-SQZ-Spleno-HPV treated animals, the majority of
these CD8+ T cells (>80%) were specific for the E7 antigen as
determined by tetramer staining compared to control mice (<5%)
and mice receiving peptide vaccine (<40%) (FIG. 36C). As seen in
FIG. 36D, administration of M-SQZ-Spleno-HPV led to regression of
tumor volume beginning 4 days post-immunization, as compared to
untreated mice or mice treated with a peptide vaccine, where the
tumor growth was not inhibited. Taken together, these data
demonstrate that immunization with M-SQZ-Spleno-HPV in the TC-1
mouse tumor model led to a significant increase in E7-specific CD8+
T cells infiltrating the tumor. The increase in E7-specific CD8+ T
cells (FIGS. 36A-C) coupled with the decrease in tumor volume (FIG.
36D) supported that M-SQZ-Spleno-HPV reduced tumor burden by
expanding E7-specific effector CD8+ T cells.
Example 37
[0965] The objective of this study is to demonstrate the
scalability of SQZ-loading of human PBMCs. Human PBMCs were
subjected to SQZ-processing in the presence of Dextran at a
manufacturing scale, and the payload delivery and PBMC viability
was assessed.
Methods
[0966] Leukopaks (HEMACARE.RTM.) containing peripheral blood of
healthy HLA-A2+ donors were obtained and PBMCs were isolated via
elutriation. The resulting PBMCs were resuspended in 120 mL RPMI to
obtain a PBMC suspension with a concentration of
7.20.times.10.sup.7 cells/mL. The RBC suspension was then either
(i) incubated, or (ii) subjected to SQZ-processing (50 psi, 4 .mu.m
diameter constriction, 2-8.degree. C.), in the presence of
fluorescent Dextran (3 kDa Dextra AF680). SQZ-processing was
subsequently quenched with placing the SQZ-processed cells into
1000 mLs of RPMI. 2 hours subsequent to incubation or
SQZ-processing, the viability as well as fluorescent Dextran
delivery were measured via flow cytometry for PBMC, and for each
cell type within: B cells (CD20+), T cells (CD3+), NK cells (CD56+)
and monocytes (CD14+).
Results
[0967] FIG. 37A shows the number of cells SQZ-processed in the
manufacturing setting described in this example is more than 3
orders of magnitude higher the number of cells SQZ-processed in an
experimental setting. As shown in FIG. 37B, there was no
significant difference in viability between the incubation control
and the SQZ-processed PBMCs, with both registering above 80%
viability. As shown in FIG. 37C, 3 kDa Dextran was delivered via
SQZ-processing into about 80% of all PBMCs, and at least into 60%
of each cell type. Over 90% of CD14+ monocytes were loaded with
Dextran after SQZ-processing. In contrast, less than 10% of PBMCs
incubated with Dextran showed any delivery. Taken together, the
results show that SQZ-mediated delivery can be used at a
manufacturing scale to deliver payloads efficiently into all
component cell types in the PBMC without any significant loss of
viability.
Example 38
[0968] In order to determine whether mRNA introduced by SQZ
delivery could be translated and expressed in PBMC subsets, human
PBMCs were subjected to SQZ-processing in the presence of mRNA
encoding CD86 or IFN.alpha.2 and the protein expression of CD86 or
IFN.alpha.2 was assessed by flow cytometry or intracellular
staining.
Methods
[0969] Human PBMCs were either left untreated (NC), or subjected to
SQZ-processing in the presence of mRNA encoding CD86 or IFN.alpha.2
(SQZ) or an empty payload (Empty SQZ). Subsequent to SQZ-loading,
the PBMCs loaded with CD86-encoding mRNA were analyzed for CD86
surface expression by flow cytometry in component cell types of B
cells (CD19+), T cells (CD3+), NK cells (CD56+) and monocytes
(CD14+). For PBMCs SQZ-processed with IFN.alpha.2-encoding mRNA,
the loaded PBMCs were incubated with GOLGIPLUG/GOLGISTOP for 4
hours to stop secretion, and subsequently analyzed for IFN.alpha.2
expression by intracellular staining in in component cell types of
B cells (CD19+), T cells (CD3+), NK cells (CD56+) and monocytes
(CD14+).
Results
[0970] As shown in FIGS. 38A and 38B, all PBMC subset populations
demonstrated expression of CD86 or IFN.alpha.2, in at least about
40% of the respective cells, following delivery of the respective
encoding mRNAs, as compared to untreated PBMCs, or control PBMCs
SQZ-processed with empty payload. CD14+ monocytes inherently
express CD86. The results indicated mRNAs can be introduced by SQZ
delivery into PBMCs for efficient expression of the encoded
protein.
Example 39
[0971] In order to determine the variation in degree and duration
in expression of candidate mRNAs introduced by SQZ delivery, human
PBMCs were SQZ-loaded with mRNA encoding CD86 or 4-1BBL and the
corresponding protein expression of CD86 or 4-1BBL was assessed by
flow cytometry.
Methods
[0972] Human PBMCs were subjected to SQZ-processing in the presence
of mRNA encoding CD86 or IFN.alpha.2 (SQZ) or an empty payload
(Empty SQZ). Subsequent to SQZ-loading, the PBMCs loaded with the
respective mRNAs were incubated for 4 hours at 37.degree. C., and
the expression of the respective encoded protein was measured every
24 hours by flow cytometry in component cell types of B cells
(CD19+), T cells (CD3+), NK cells (CD56+) and monocytes
(CD14+).
Results
[0973] FIGS. 39A and 39B shows the expression of CD86 and 4-1BBL in
the T cell subset populations within the PBMCs SQZ-loaded with the
respective mRNAs. As shown in FIG. 39A, The T cell subset within
PBMCs loaded with CD86-encoding mRNA showed a significant increase
in the percentage of CD86+ cells compared to control, at 4 hours to
48 hours post SQZ-processing, and the percentage of CD86+ cells
slightly decreased only at 72 hours post SQZ processing. As shown
in FIG. 39B, The T cell subset within PBMCs loaded with
4-1BBL-encoding mRNA showed a moderate increase in the percentage
of 4-1BBL+ cells at 4 hours post SQZ-processing, and the percentage
of 4-1BBL+-positive cells noticeably decreased after only 24 hours
of incubation. The results indicated that SQZ-delivery of different
candidate mRNAs can result in different degrees and durations in
the expression of encoded protein.
Example 40
[0974] In order to determine whether translation-enhancing
modifications of mRNA will affect expression of candidate mRNAs
introduced by SQZ delivery, human PBMCs were SQZ-loaded with an
unmodified eGFP-encoding mRNA or a GFP-endocing mRNA with
5-metoxyuridine backbone (5moU), and the corresponding eGFP
expression was assessed by flow cytometry.
Methods
[0975] Human PBMCs were subjected to SQZ-processing in the presence
of 0 to 200 .mu.g/mL of an unmodified eGFP-encoding mRNA or a
GFP-endocing mRNA with 5moU backbone. Subsequent to SQZ-loading,
the PBMCs loaded with the respective mRNAs were incubated for 4
hours at 37.degree. C., and the eGFP expression was measured by
mean fluorescence intensity (MFI) via flow cytometry in component
cell types of B cells (CD19+), T cells (CD3+), NK cells (CD56+) and
monocytes (CD14+).
Results
[0976] FIG. 40 shows the expression of eGFP in the T cell subset
populations within the PBMCs SQZ-loaded with the unmodified eGFP
mRNA or the 5moU-modified eGFP mRNA. As shown in FIG. 40, The T
cell subset within PBMCs loaded with unmodified eGFP mRNA showed a
higher MFI compared to that loaded with 5moU-modified eGFP mRNA.
The results indicated that 5moU RNA modification did not result in
enhanced translation of mRNAs delivered by SQZ processing.
Example 41
[0977] In order to determine whether cytokines encoded by mRNAs
introduced by SQZ delivery could be translated, expressed and
secreted in PBMC subsets, human PBMCs were SQZ-loaded with mRNA
encoding IL-12, IFN.alpha. or IL-2 respectively and the
corresponding secretion of IL-12, IFN.alpha. or IL-2 was assessed
by ELISA.
Methods
[0978] Human PBMCs were either left untreated (NC), or subjected to
SQZ-processing in the presence of mRNA encoding IL-12 (50 .mu.g/mL
IL-12a and 50 .mu.g/mL IL-12b mRNA), mRNA encoding IFN.alpha. (100
.mu.g/mL) or mRNA encoding IL-2 (SQZ) (100 .mu.g/mL), or an empty
payload (Empty SQZ). After SQZ-loading, the respective PBMCs loaded
with the cytokine-encoding mRNAs were then incubated for 4 hours at
37.degree. C., and subsequently the supernatant was assayed by
ELISA to determine the expression and secretion of the respective
cytokines.
Results
[0979] As shown in FIG. 41, PBMCs SQZ-loaded with cytokine-encoding
mRNAs exhibited significant expression and secretion of IL-12,
IFN.alpha. or IL-2 respectively. The results indicated mRNAs can be
introduced by SQZ delivery into PBMCs for efficient expression and
secretion of encoded cytokines.
Example 42
[0980] As illustrated in FIG. 42A, in addition to T cell receptor
engagement by antigen presenting cells (Signal 1), the activation
of an immune response is enhanced by additional signals such as
co-stimulatory receptor activation (Signal 2) and cytokine receptor
binding (Signal 3). In order to determine whether mRNAs introduced
by SQZ delivery could be translated and expressed into protein
effectors for these enhancing signals, human PBMCs were SQZ-loaded
with mRNA encoding CD70 or 4-1BBL (for Signal 2) and/or with mRNA
encoding IFN.alpha.2 or IL-2 (Signal 3) respectively. The
corresponding expression of CD70 or 4-1BBL was measured by flow
cytometry, while the corresponding secretion of IFN.alpha.2 or IL-2
was measured from culture supernatant via ELISA.
Methods
[0981] Human PBMCs were either left untreated (NC), or subjected to
SQZ-processing in the presence of respective mRNA encoding CD70,
4-1BBL, IFN.alpha.2 or IL-2. Subsequent to SQZ-processing with mRNA
encoding CD70 or 4-1BBL, the PBMCs loaded with the respective mRNAs
were incubated for 4 hours at 37.degree. C., and the expression of
the respective encoded protein CD70 or 4-1BBL was measured every 24
hours by flow cytometry in component cell types of B cells (CD19+),
T cells (CD3+), NK cells (CD56+) and monocytes (CD14+). After
SQZ-loading with mRNA encoding IFN.alpha.2 or IL-2, the PBMCs
loaded with the respective mRNAs were then incubated for 4 hours at
37.degree. C., and subsequently the supernatants were collected at
time points up to 24 hours, and assayed by ELISA to determine the
expression and secretion of the respective cytokines IFN.alpha.2 or
IL-2.
Results
[0982] As shown in FIG. 42B, all loaded PBMC subset populations
exhibited expression of CD70 or 4-1BBL respectively, following
delivery of the respective encoding mRNAs, as shown by the
increased in mean fluorescence intensity assayed by flow cytometry.
The respective expression of CD70 or 4-1BBL was maintained for at
least 48 hours. Among the PBMC subsets, monocytes exhibited higher
expression of the SQZ-delivered mRNA encoding CD70 or 4-1BBL. As
shown in FIG. 42C, PBMCs SQZ-loaded with cytokine-encoding mRNAs
exhibited significant expression and secretion of IFN.alpha.2 or
IL-2 respectively, and the expression and secretion was maintained
for at least 24 hours post-SQZ processing. The results indicated
mRNAs can be introduced by SQZ delivery into PBMCs for efficient
expression and secretion of the molecules providing enhancing
signals in immune activation.
Example 43
[0983] As illustrated in FIG. 42A, in addition to T cell receptor
engagement by antigen presenting cell (Signal 1), the activation of
an immune response is enhanced by additional signals such as
co-stimulatory receptor activation (Signal 2) and cytokine receptor
binding (Signal 3). In order to determine whether Signal 1
activation will affect translation and expression of mRNAs
introduced by SQZ delivery, crafted mouse splenocytes were
SQZ-loaded with candidate mRNA (eGFP or CD86) with or without
stimulation of Concanavalin A (ConA), an antigen-independent
mitogen inducing Signal 1. The corresponding eGFP and CD86
expression was then measured by flow cytometry.
Methods
[0984] Splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation, leading to a
splenocyte composition more representative of human PBMCs (i.e.,
crafted splenocytes). The crafted murine splenocytes were either
left unstimulated (No ConA) or stimulated with ConA before or after
SQZ processing (pre-SQZ stim or post-SQZ stim). For SQZ-mediated
delivery, the crafted murine splenocytes were subjected to
SQZ-processing in the presence of respective mRNA encoding eGFP or
CD86 (at 100 .mu.g/mL) or an empty payload (Empty SQZ). Subsequent
to SQZ-processing with mRNA encoding eGFP or CD86, the crafted
murine splenocytes loaded with the respective mRNAs were incubated
for 4 hours at 37.degree. C., and the expression of the respective
encoded protein eGFP or CD86 was measured by flow cytometry in
component cell types of B cells (CD19+), T cells (CD3+), NK cells
(CD56+) and monocytes (CD14+).
Results
[0985] As shown in FIG. 43A, crafted murine splenocytes without
ConA stimulation or stimulated by ConA after SQZ-loading showed
moderate eGFP translation and expression, with 15.3% to 17.0% of
the T cell subset being GFP+ in flow analysis. Stimulation of
crafted murine splenocytes with ConA prior to SQZ-loading
dramatically increased eGFP translation and expression, with 91.1%
of the T cell subset being GFP+ in flow analysis. Similarly, as
shown in FIG. 43B, crafted murine splenocytes without ConA
stimulation showed moderate CD86 translation and expression, as
demonstrated by the small increase in CD86 MFI in the T cell subset
population compared to control, whereas stimulation of crafted
murine splenocytes with ConA prior to SQZ-loading dramatically
increased CD86 translation and expression, as demonstrated by the
significant increase in CD86 MFI in the T cell subset population
compared to control. These results indicated that ConA stimulation
enhances translation of candidate mRNAs delivered by SQZ
processing.
Example 44
[0986] As illustrated in FIG. 42A, in addition to T cell receptor
engagement by antigen presenting cell (Signal 1), the activation of
an immune response is enhanced by additional signals such as
co-stimulatory receptor activation (Signal 2) and cytokine receptor
binding (Signal 3). In order to determine the expression efficacy
and kinetics of mRNAs encoding Signal 2 and Signal 3 effectors in
murine cells, crafted murine splenocytes were SQZ-loaded with
candidate mRNAs (CD70, CD80, CD86 and OX40L for Signal 2; IL-12,
IL-2 and IFN.alpha.2 for Signal 3). The corresponding expression of
CD70, CD80, CD86 or OX40L was measured by flow cytometry, while the
corresponding secretion of IL-12, IL-2 and IFN.alpha.2 was measured
from culture supernatant via ELISA.
Methods
[0987] Splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation, leading to a
splenocyte composition more representative of human PBMCs (i.e.,
crafted splenocytes). For SQZ-mediated delivery, the crafted murine
splenocytes were subjected to SQZ-processing in the presence of
respective mRNA encoding CD70, CD80, CD86 or OX40L, or mRNA
encoding IL-12, IL-2 and IFN.alpha.2 (all at 100 .mu.g/mL); or an
empty payload (Empty SQZ). Subsequent to SQZ-processing with mRNA
encoding Signal 2 effectors, the crafted murine splenocytes loaded
with the respective mRNAs were incubated for 4 hours at 37.degree.
C., and the expression of the respective encoded protein CD70,
CD80, CD86 or OX40L was measured by flow cytometry for 48 hours in
component cell types of B cells (CD19+), T cells (CD3+), NK cells
(CD56+) and monocytes (CD14+). Subsequent to SQZ-processing with
mRNA encoding Signal 2 effectors, the crafted murine splenocytes
loaded with the respective mRNAs were incubated for 4 hours at
37.degree. C., and the expression of the respective encoded protein
CD70, CD80, CD86 or OX40L was measured by flow cytometry for 48
hours in component cell types of B cells (CD19+), T cells (CD3+),
NK cells (CD56+) and monocytes (CD14+). After SQZ-processing with
mRNA encoding Signal 3 effectors (cytokines), the crafted murine
splenocytes loaded with the respective mRNAs were then incubated
for 4 hours at 37.degree. C., and subsequently the supernatants
were collected at time points up to 48 hours, and assayed to
determine the expression and secretion of the respective cytokines
IL-12, IL-2 and IFN.alpha.2 (FIG. 44A).
Results
[0988] As shown in FIG. 44B, crafted murine splenocytes SQZ-loaded
with Signal 2 effectors (CD70, CD80, CD86 or OX40L) showed
significant translation and expression of the respective proteins,
as illustrated by the appreciable increase in MFI respectively.
CD86 expression was observed to persist up to at least 48 hours
post-SQZ, CD70 expression dissipated at 48 hours post-SQZ, whereas
CD80 and OX40K dissipated at 24 hours post-SQZ. As shown in FIG.
44C, crafted murine splenocytes SQZ-loaded with Signal 3 effectors
(IL-12, IL-2 or IFN.alpha.2) showed significant expression and
secretion of the respective cytokines, as illustrated by the
appreciable increase in signal detected in ELISA assay. IL-12 and
IL-2 secretion was significantly induced at 4 hours post-SQZ,
increased slightly at 24 hours post-SQZ and slightly tapered off at
48 hours. In contrast, IFN.alpha.2 secretion significantly
increased from 4 hours post-SQZ to 24 hours post-SQZ and further
increased at 48 hours post-SQZ. These results indicated that mRNAs
encoding Signal 2 and Signal 3 effectors were effectively
translated and expressed following SQZ-mediated intracellular
delivery. However, the duration and magnitude of expression of
these effectors varied by the effector molecule.
Example 45
[0989] In order to determine whether mRNAs encoding Signal 2 and
Signal 3 effectors in murine cells can enhance antigen-specific
immune response in vitro, ConA-activated crafted murine splenocytes
were SQZ-loaded with OVA antigen as well as candidate mRNAs (CD70,
CD80, CD86 for Signal 2; IL-2 for Signal 3). The SQZ-loaded crafted
splenocytes were subsequently co-cultured with OVA-specific OT-I
CD8+ T cells, and the activation of OT-I T cells was measured via
ELISA of IFN-.gamma. secretion.
Methods
[0990] Splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation, leading to a
splenocyte composition more representative of human PBMCs (i.e.,
crafted splenocytes), and subsequently activated with ConA. For
SQZ-mediated delivery, the crafted murine splenocytes were
subjected to SQZ-processing in the presence of (i) OVA (5 .mu.g/mL)
as well as (ii) respective mRNA encoding CD70, CD80, or CD86, or
mRNA encoding IL-12 (all at 100 .mu.g/mL); or without mRNA.
Subsequent to SQZ-processing with mRNA encoding Signal 2 or Signal
3 effectors, the crafted murine splenocytes loaded with the
respective mRNAs were incubated for 4 hours at 37.degree. C., and
the co-cultured for 24 hours with OT-I CD8+ T cells at a 1:2 or 1:4
ratio. To assay T cell activation, supernatant was collected from
the co-culture and subjected to IFN-.gamma. ELISA assay (FIG.
45A).
Results
[0991] As shown in FIGS. 45B and 45C, ConA-activated, crafted
murine splenocytes SQZ-loaded with OVA combined with Signal 2
effectors (CD70, CD80, or CD86) or Signal 3 effector (IL-12) showed
significant increase in activation of OVA-specific OT-I CD8+ T
cells. The increase in OVA-specific T cell response was
particularly significant when CD86 mRNA or IL-12 mRNA was
co-delivered with OVA into the crafted murine splenocytes. These
results indicated that in addition to being capable of being
translated and expressed, the mRNAs encoding Signal 2 and Signal 3
effectors could enhance the ability of murine splenocytes' function
as antigen presenting cells in activating antigen-specific T cell
response.
Example 46
[0992] In order to determine whether mRNAs encoding antigens in
human PBMCs can enhance antigen-specific immune response in vitro,
human PBMCs were SQZ-loaded with a variety of available antigens,
and subsequently co-cultured with the respective antigen-specific
ASTARTE responder T cells, where the activation of responder T
cells was measured via ELISA of IFN-.gamma. secretion.
Methods
[0993] Human PBMCs were either (i) left untreated (NC), (ii) pulsed
with 1 .mu.g/mL of respective peptide antigen (PP, positive
control) or (iii) subjected to SQZ-processing in the presence of
100 .mu.g/mL mRNA encoding respective antigen (E7, HSV GF1, MART-1,
pp65 or Influenza M1), or (iv) SQZ-processed with an empty payload
(Empty SQZ). Western blot was used to analyze translation of the
loaded mRNAs at 4 hours and 24 hours after SQZ-processing. For
assaying immune activation, after SQZ-loading, the PBMCs loaded
with the respective antigen-encoding mRNAs were incubated for 4
hours at 37.degree. C., and co-cultured for 24 hours with
respective antigen-specific ASTARTE responder T cells. To measure
for T cell activation, supernatant was collected from the
co-culture and subjected to IFN-.gamma. ELISA.
Results
[0994] As shown in FIGS. 46B and 46C, subsequent to SQZ-mediated
delivery of mRNAs encoding HPV16-E7 and Influenza M1, the
respective antigens were translated and expressed, as indicated by
the clear bands on Western blots. For T cell activation assays
(FIG. 46A), PBMCs SQZ-loaded with mRNAs encoding E7, HSV GD1,
MART-1 or pp65 antigens did not induce observable responder T cell
activation compared to PBMCs subjected to peptide pulse (PP), as
demonstrated by lack of IFN-.gamma. secretion after co-culture; but
PBMCs SQZ-loaded with mRNA encoding Influenza M1 strongly induced
M1-specific T cell activation, as observed by significant induction
of IFN-.gamma. secretion after co-culture as compared to PBMC
subjected to peptide pulse (PP). These results indicated that,
using current protocols, antigen-encoding mRNAs could be employed
in SQZ-mediated delivery to facilitate antigen presentation and
stimulate antigen-specific T cell response for some but not all
antigens.
Example 47
[0995] In order to compare the efficacy of SQZ-delivered antigen
encoding mRNAs and peptide antigens in facilitating murine immune
cells to activate an antigen-specific response in vitro, whole
murine splenocytes were SQZ-loaded with OVA proteins or
OVA-encoding mRNA and subsequently co-cultured with the
OVA-specific OT-I CD8+ T cells, where the activation of OT-I T
cells was measured via CD69 expression.
Methods
[0996] Splenocytes were obtained from spleens of female C57BL/6J
donor mice, and were subjected SQZ-processing using a 3.5 .mu.m
width, 30 .mu.m height constriction at 60 psi under room
temperature, in the presence of (i) mRNA encoding OVA (0 to 250
nM), or OVA protein (0 to 1250 nM). Western blot was used to
analyze translation of the loaded mRNAs at 4 hours and 24 hours
after SQZ-processing. For assaying immune activation, after
SQZ-loading, the splenocytes loaded with the respective
OVA-encoding mRNA or with OVA protein were incubated for 4 hours at
37.degree. C., and co-cultured for 24 hours with OT-I CD8+ T cells.
To measure for T cell activation, CD69 expression on OT-I CD8+ T
cells was measured via flow cytometry (FIG. 47A).
Results
[0997] As shown in FIG. 47B, when SQZ-loaded at the same respective
molarity in murine splenocytes, OVA-encoding mRNA was much more
potent (.about.20 fold more potent) than OVA proteins in
facilitating the activation of OT-I CD8+ T cells. Splenocytes
SQZ-processed in the presence of <50 nM OVA mRNA led to a
significant percentage of OT-I CD8+ T cells expressing CD69 upon
co-culture. In comparison, splenocytes SQZ-processed in the
presence of at least 1000 nM OVA protein led to achieve similar
percentages of CD69-expressing OT-I CD8+ T cells upon co-culture.
These results indicated that at least for OVA antigen, splenocyte
loading of antigen-encoding mRNAs was more effective than that of
protein in facilitating in vitro activation of antigen-specific T
cell response.
Example 48
[0998] In order to determine the effect of combination with immune
checkpoint inhibitors on the ability of antigen-loaded splenocytes
on tumor growth inhibition in a therapeutic setting, mice implanted
with HPV E7-expressing TC1 tumor model were either administered
with anti-CTLA4 injections, with crafted murine splenocytes
SQZ-loaded with E7 SLP (M-SQZ-Spleno-HPV), or administered with a
combination of both, and the tumor volumes and survival were
plotted against time.
Methods
[0999] At Day 0, C57BL/6J female mice were injected in the right
rear flank with TC1 tumor cells (50 k cells/mouse). On Day 10
(prime), splenocytes were obtained from spleens of female C57BL/6J
donor mice, and combined with splenocytes that have had their B
cells depleted by negative immunomagnetic separation to better
mimic human PBMCs, leading to a splenocyte composition more
representative of human PBMCs (i.e., crafted splenocytes). Crafted
splenocytes were SQZ-loaded with 20 .mu.M E7 SLP
(GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR (SEQ ID NO: 25)), incubated
with CpG 1826 (1 .mu.M in R10) for 4 hours at 37.degree. C. and the
loaded crafted murine splenocytes were injected IV
(retro-orbitally) into the tumor bearing mice (M-SQZ-PBMC-HPV)
(1.times.10.sup.6 cells per mice). Cohorts of mice having received
the loaded splenocytes and mice without the loaded splenocytes were
then administered with anti-CTLA4 injections at the indicated
schedules (Sch.1: Day 11, 14, 17; Sch.2: Day 17, 20, 24; Sch.3: Day
24, 28, 31 after TC-1 implantation). Control mice were left
untreated. (10 mice per group) (FIGS. 48A, 48B). TC-1 tumor growth
was measured beginning 1 week post-tumor implantation two times per
week and compared to tumor growth in untreated mice for 60
days.
Results
[1000] Tumor growth, as measured by the formula
((length.times.width.sup.2)/2), was compared between control mice
(Untreated) and groups treated with crafted E7-loaded splenoyctes
(M-SQZ-PBMC-HPV), groups treated with anti-CTLA4 (.alpha.-CTLA4)
and groups treated with a combination of both
(M-SQZ-PBMC-HPV+.alpha.-CTLA4). As shown in FIG. 48C, while there
was no observable difference between the tumor growth in control
mice and in mice treated with anti-CTLA4 alone (Sch. 1
.alpha.-CTLA4; Sch. 2 .alpha.-CTLA4; Sch. 3 .alpha.-CTLA4), there
was an appreciable inhibition in the rate of tumor growth for mice
primed with E7-loaded splenocytes (M-SQZ-PBMC-HPV). Noticeably, the
combination of E7-loaded splenocytes and anti-CTLA4 administration
showed a significant additive effect in inhibiting TC-1 tumor
growth (M-SQZ-PBMC-HPV+.alpha.-CTLA4) (FIG. 48C).
[1001] Untreated mice, mice treated with anti-CTLA4 alone (Sch. 1
.alpha.-CTLA4; Sch. 2 .alpha.-CTLA4; Sch. 3 .alpha.-CTLA4) or mice
primed with E7-loaded splenocytes (M-SQZ-PBMC-HPV) all developed
tumor at Day 40 or earlier (FIG. 48D). In comparison, mice
receiving combination of E7-loaded splenocytes and anti-CTLA4
administration (M-SQZ-PBMC-HPV+.alpha.-CTLA4) showed inhibited or
delayed tumor development, with 2, 1, and 3 mice being tumor free
at Day 60 for M-SQZ-PBMC-HPV+Sch.1 .alpha.-CTLA4,
M-SQZ-PBMC-HPV+Sch.2 .alpha.-CTLA4 and M-SQZ-PBMC-HPV+Sch.3
.alpha.-CTLA4 respectively (FIG. 48E).
[1002] Consistent with the results in tumor growth inhibition, the
combination of E7-loaded splenocytes and anti-CTLA4 administration
also showed an additive effect in improving survival of TC-1
carrying mice. Untreated mice showed a median survival of 38 days,
whereas mice treated with anti-CTLA4 alone (Sch. 1 .alpha.-CTLA4;
Sch. 2 .alpha.-CTLA4; Sch. 3 .alpha.-CTLA4) showed median survival
of 32, 33 and 35.5 days respectively. Mice primed with E7-loaded
splenocytes (M-SQZ-PBMC-HPV) showed slightly improved median
survival at 50 days. Noticeably, median survival has not been
reached for mice receiving combinations of E7-loaded splenocytes
and anti-CTLA4 administration (M-SQZ-PBMC-HPV+.alpha.-CTLA4) at Day
60 subsequent to TC-1 implantation (FIG. 48F).
[1003] These results indicated that therapeutic combination of
antigen-loaded splenocytes and immune checkpoint inhibitor afforded
additive benefits in inhibiting tumor growth and in improving
survival.
TABLE-US-00002 Sequence Listing SEQ ID NO Sequence Description 1
TIHDIILECV HPV16-E6 (29-38), human epitope 2 EVYDFAFRDL HPV16-E6
(48-57), murine epitope 3 YMLDLQPETT HPV16-E7 (11-20), human
epitope 4 RAHYNIVTF HPV16-E7 (49-57), murine epitope 5 LPQLSTELQT
HPV16-E6 (19-28) N-terminal polypeptide, human 6 QLCTELQT HPV16-E6
(21-28) N-terminal polypeptide, human 7 KQQLLRR HPV16-E6 (41-47)
N-terminal polypeptide, native murine 8 VYSKQQLLRR HPV16-E6 (38-47)
N-terminal polypeptide, classic murine 9 MHGDTPTLHE HPV16-E7 (1-10)
N-terminal polypeptide, human 10 GQAEPD HPV16-E7 (43-48) N-terminal
polypeptide, murine 11 YSKQQLLRREVYDFAF HPV16-E6 (39-54) C-terminal
polypeptide, human 12 YCKQQLL HPV16-E6 (39-45) C-terminal
polypeptide, human 13 CIVYRDGN HPV16-E6 (58-65) C-terminal
polypeptide, native murine 14 SIVYRDGNPYAVSDK HPV16-E6 (58-72)
C-terminal polypeptide, classic murine 15 DLYCYEQLNDSSEEE HPV16-E7
(21-35) C-terminal polypeptide, human 16 CCKCDSTLRLCVQSTHVDIR
HPV16-E7 (58-77 C-terminal polypeptide, native murine 17
SSKSDSTLRLSVQSTHVDIR HPV16-E7 (58-77) C-terminal polypeptide,
classic murine 18 LPQLSTELQTTIHDIILECVYSKQQLLRREVYDFAF HPV16-E6
(19-54) SLP, human 19 QLCTELQTTIHDIILECVYCKQQLL HPV16-E6 (21-45)
SLP, human 20 KQQLLRREVYDFAFRDLCIVYRDGN HPV16-E6 (41-65) SLP,
native murine 21 VYSKQQLLRREVYDFAFRDLSIVYRDGNPYAVSDK HPV16-E6
(38-72) SLP, classic murine 22 MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE
HPV16-E7 (1-35) SLP, human 23 QLCTELQTYMLDLQPETTYCKQQLL HPV16-E7.6
SLP, human 24 GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR HPV16-E7 (43-77)
SLP, native murine 25 GQAEPDRAHYNIVTFSSKSDSTLRLSVQSTHVDIR HPV16-E7
(43-77) SLP, classic murine 26 ggGGTCAACGTTGAgggggg ODN 1585 Bases
shown in capital letters are (Class A, phosphodiester, and those
mouse-specific) in lower case are phosphorothioate 27
ggGGGACGA:TCGTCgggggg ODN 2216 Bases shown in capital letters are
(Class A, phosphodiester, and those human-selective) in lower case
are phosphorothioate 28 gggGACGAC:GTCGTGgggggg ODN 2336 Bases shown
in capital letters are (Class A, phosphodiester, and those human
preferred) in lower case are phosphorothioate 29
tccatgacgttcctgatgct ODN 1668 Bases shown in capital letters are
(Class B, phosphodiester, and those mouse specific) in lower case
are phosphorothioate 30 tccatgacgttcctgacgtt ODN 1826 Bases are
phosphorothioate (Class B, mouse specific) 31
tcgtcgttttgtcgttttgtcgtt ODN 2006 Bases are phosphorothioate (Class
B, human selective) 32 tcg tcg ttg tcg ttt tgt cgt t ODN 2007 Bases
are phosphorothioate (Class B, bovine/porcine) 33 tcg acg ttc gtc
gtt cgt cgt tc ODN BW006 Bases are phosphorothioate (Class B, human
& mouse) 34 tcg cga cgt tcg ccc gac gtt cgg ta ODN D-SL01 Bases
are phosphorothioate (Class B, multispecies) 35
tcgtcgttttcggcgc:gcgccg ODN 2395 Bases are phosphorothioate (Class
C, human/mouse) 36 tcgtcgtcgttc:gaacgacgttgat ODN M362 Bases are
phosphorothioate (Class C, human/mouse) 37 tcg cga acg ttc gcc gcg
ttc gaa cgc gg ODN D-SL03 Bases are phosphorothioate (Class C,
multispecies) 38 MHGDTPTLHEYMLDLQPETTDLYCYEQLNDSSEEE E7 39
LYCYEQLNDSSEEEDEIDGPAGQAEPDRAHYNIVT E7 40
GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIR E7 41
TLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP E7 42
MHQKRTAMFQDPQERPRKLPQLCTELQTTIHD E6 43
LPQLCTELQTTIHDIILECVYCKQQLLRREVY E6 44 KQQLLRREVYDFAFRDLCIVYRDGN E6
45 RDLCIVYRDGNPYAVCDKCLKFYSKI E6 46 DKCLKFYSKISEYRHYCYSLYGTTL E6 47
HYCYSLYGTTLEQQYNKPLCDLLIR E6 48 YGTTLEQQYNKPLCDLLIRCINCQKPLCPEEK E6
49 RCINCQKPLCPEEKQRHLDKKQRFHNIRGRWT E6 50
DKKQRFHNIRGRWTGRCMSCCRSSRTRRETQL E6 51
PPWQAGILARNLVPMVATVQGQNLKYQEFFWDAND pp65 SLP, human 52 PPWQAGILAR
pp65 N-terminal polypeptide, human 53 QGQNLKYQEFFWDAND pp65
C-terminal polypeptide, human
Sequence CWU 1
1
55110PRTArtificial SequenceSynthetic Construct 1Thr Ile His Asp Ile
Ile Leu Glu Cys Val1 5 10210PRTArtificial SequenceSynthetic
Construct 2Glu Val Tyr Asp Phe Ala Phe Arg Asp Leu1 5
10310PRTArtificial SequenceSynthetic Construct 3Tyr Met Leu Asp Leu
Gln Pro Glu Thr Thr1 5 1049PRTArtificial SequenceSynthetic
Construct 4Arg Ala His Tyr Asn Ile Val Thr Phe1 5510PRTHomo sapiens
5Leu Pro Gln Leu Ser Thr Glu Leu Gln Thr1 5 1068PRTHomo sapiens
6Gln Leu Cys Thr Glu Leu Gln Thr1 577PRTMus musculus 7Lys Gln Gln
Leu Leu Arg Arg1 5810PRTMus musculus 8Val Tyr Ser Lys Gln Gln Leu
Leu Arg Arg1 5 10910PRTHomo sapiens 9Met His Gly Asp Thr Pro Thr
Leu His Glu1 5 10106PRTMus musculus 10Gly Gln Ala Glu Pro Asp1
51116PRTHomo sapiens 11Tyr Ser Lys Gln Gln Leu Leu Arg Arg Glu Val
Tyr Asp Phe Ala Phe1 5 10 15127PRTHomo sapiens 12Tyr Cys Lys Gln
Gln Leu Leu1 5138PRTMus musculus 13Cys Ile Val Tyr Arg Asp Gly Asn1
51415PRTMus musculus 14Ser Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala
Val Ser Asp Lys1 5 10 151515PRTHomo sapiens 15Asp Leu Tyr Cys Tyr
Glu Gln Leu Asn Asp Ser Ser Glu Glu Glu1 5 10 151620PRTMus musculus
16Cys Cys Lys Cys Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His1
5 10 15Val Asp Ile Arg 201720PRTMus musculus 17Ser Ser Lys Ser Asp
Ser Thr Leu Arg Leu Ser Val Gln Ser Thr His1 5 10 15Val Asp Ile Arg
201836PRTHomo sapiens 18Leu Pro Gln Leu Ser Thr Glu Leu Gln Thr Thr
Ile His Asp Ile Ile1 5 10 15Leu Glu Cys Val Tyr Ser Lys Gln Gln Leu
Leu Arg Arg Glu Val Tyr 20 25 30Asp Phe Ala Phe 351925PRTHomo
sapiens 19Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile
Leu Glu1 5 10 15Cys Val Tyr Cys Lys Gln Gln Leu Leu 20 252025PRTMus
musculus 20Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe
Arg Asp1 5 10 15Leu Cys Ile Val Tyr Arg Asp Gly Asn 20 252135PRTMus
musculus 21Val Tyr Ser Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr Asp
Phe Ala1 5 10 15Phe Arg Asp Leu Ser Ile Val Tyr Arg Asp Gly Asn Pro
Tyr Ala Val 20 25 30Ser Asp Lys 352235PRTHomo sapiens 22Met His Gly
Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp Leu Gln1 5 10 15Pro Glu
Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn Asp Ser Ser 20 25 30Glu
Glu Glu 352325PRTHomo sapiens 23Gln Leu Cys Thr Glu Leu Gln Thr Tyr
Met Leu Asp Leu Gln Pro Glu1 5 10 15Thr Thr Tyr Cys Lys Gln Gln Leu
Leu 20 252435PRTMus musculus 24Gly Gln Ala Glu Pro Asp Arg Ala His
Tyr Asn Ile Val Thr Phe Cys1 5 10 15Cys Lys Cys Asp Ser Thr Leu Arg
Leu Cys Val Gln Ser Thr His Val 20 25 30Asp Ile Arg 352535PRTMus
musculus 25Gly Gln Ala Glu Pro Asp Arg Ala His Tyr Asn Ile Val Thr
Phe Ser1 5 10 15Ser Lys Ser Asp Ser Thr Leu Arg Leu Ser Val Gln Ser
Thr His Val 20 25 30Asp Ile Arg 352620DNAArtificial
SequenceSynthetic ConstructMISC_FEATURE1, 2,
(15)..(20)Phosphorothioate basesMISC_FEATURE(3)..(14)Phosphodiester
bases 26ggggtcaacg ttgagggggg 202720DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE1, 2, (15)..(20)Phosphorothioate
basesMISC_FEATURE(3)..(14)Phosphodiester bases 27gggggacgat
cgtcgggggg 202821DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(3), (15)..(20)Phosphorothioate
basesMISC_FEATURE(4)..(14)Phosphodiester bases 28ggggacgacg
tcgtgggggg g 212920DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(20)Phosphorothioate bases 29tccatgacgt
tcctgatgct 203020DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(20)Phosphorothioate bases 30tccatgacgt
tcctgacgtt 203124DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(24)Phosphorothioate bases 31tcgtcgtttt
gtcgttttgt cgtt 243222DNABos
taurusMISC_FEATURE(1)..(22)Phosphorothioate bases 32tcgtcgttgt
cgttttgtcg tt 223323DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(23)Phosphorothioate bases 33tcgacgttcg
tcgttcgtcg ttc 233426DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(26)Phosphorothioate bases 34tcgcgacgtt
cgcccgacgt tcggta 263522DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(22)Phosphorothioate bases 35tcgtcgtttt
cggcgcgcgc cg 223625DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(25)Phosphorothioate bases 36tcgtcgtcgt
tcgaacgacg ttgat 253729DNAArtificial SequenceSynthetic
ConstructMISC_FEATURE(1)..(29)Phosphorothioate bases 37tcgcgaacgt
tcgccgcgtt cgaacgcgg 293835PRTArtificial SequenceSynthetic
Construct 38Met His Gly Asp Thr Pro Thr Leu His Glu Tyr Met Leu Asp
Leu Gln1 5 10 15Pro Glu Thr Thr Asp Leu Tyr Cys Tyr Glu Gln Leu Asn
Asp Ser Ser 20 25 30Glu Glu Glu 353935PRTArtificial
SequenceSynthetic Construct 39Leu Tyr Cys Tyr Glu Gln Leu Asn Asp
Ser Ser Glu Glu Glu Asp Glu1 5 10 15Ile Asp Gly Pro Ala Gly Gln Ala
Glu Pro Asp Arg Ala His Tyr Asn 20 25 30Ile Val Thr
354035PRTArtificial SequenceSynthetic Construct 40Gly Gln Ala Glu
Pro Asp Arg Ala His Tyr Asn Ile Val Thr Phe Cys1 5 10 15Cys Lys Cys
Asp Ser Thr Leu Arg Leu Cys Val Gln Ser Thr His Val 20 25 30Asp Ile
Arg 354135PRTArtificial SequenceSynthetic Construct 41Thr Leu Arg
Leu Cys Val Gln Ser Thr His Val Asp Ile Arg Thr Leu1 5 10 15Glu Asp
Leu Leu Met Gly Thr Leu Gly Ile Val Cys Pro Ile Cys Ser 20 25 30Gln
Lys Pro 354232PRTArtificial SequenceSynthetic Construct 42Met His
Gln Lys Arg Thr Ala Met Phe Gln Asp Pro Gln Glu Arg Pro1 5 10 15Arg
Lys Leu Pro Gln Leu Cys Thr Glu Leu Gln Thr Thr Ile His Asp 20 25
304332PRTArtificial SequenceSynthetic Construct 43Leu Pro Gln Leu
Cys Thr Glu Leu Gln Thr Thr Ile His Asp Ile Ile1 5 10 15Leu Glu Cys
Val Tyr Cys Lys Gln Gln Leu Leu Arg Arg Glu Val Tyr 20 25
304425PRTArtificial SequenceSynthetic Construct 44Lys Gln Gln Leu
Leu Arg Arg Glu Val Tyr Asp Phe Ala Phe Arg Asp1 5 10 15Leu Cys Ile
Val Tyr Arg Asp Gly Asn 20 254526PRTArtificial SequenceSynthetic
Construct 45Arg Asp Leu Cys Ile Val Tyr Arg Asp Gly Asn Pro Tyr Ala
Val Cys1 5 10 15Asp Lys Cys Leu Lys Phe Tyr Ser Lys Ile 20
254625PRTArtificial SequenceSynthetic Construct 46Asp Lys Cys Leu
Lys Phe Tyr Ser Lys Ile Ser Glu Tyr Arg His Tyr1 5 10 15Cys Tyr Ser
Leu Tyr Gly Thr Thr Leu 20 254725PRTArtificial SequenceSynthetic
Construct 47His Tyr Cys Tyr Ser Leu Tyr Gly Thr Thr Leu Glu Gln Gln
Tyr Asn1 5 10 15Lys Pro Leu Cys Asp Leu Leu Ile Arg 20
254832PRTArtificial SequenceSynthetic Construct 48Tyr Gly Thr Thr
Leu Glu Gln Gln Tyr Asn Lys Pro Leu Cys Asp Leu1 5 10 15Leu Ile Arg
Cys Ile Asn Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys 20 25
304932PRTArtificial SequenceSynthetic Construct 49Arg Cys Ile Asn
Cys Gln Lys Pro Leu Cys Pro Glu Glu Lys Gln Arg1 5 10 15His Leu Asp
Lys Lys Gln Arg Phe His Asn Ile Arg Gly Arg Trp Thr 20 25
305032PRTArtificial SequenceSynthetic Construct 50Asp Lys Lys Gln
Arg Phe His Asn Ile Arg Gly Arg Trp Thr Gly Arg1 5 10 15Cys Met Ser
Cys Cys Arg Ser Ser Arg Thr Arg Arg Glu Thr Gln Leu 20 25
305135PRTHomo sapiens 51Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg Asn
Leu Val Pro Met Val1 5 10 15Ala Thr Val Gln Gly Gln Asn Leu Lys Tyr
Gln Glu Phe Phe Trp Asp 20 25 30Ala Asn Asp 355210PRTHomo sapiens
52Pro Pro Trp Gln Ala Gly Ile Leu Ala Arg1 5 105316PRTHomo sapiens
53Gln Gly Gln Asn Leu Lys Tyr Gln Glu Phe Phe Trp Asp Ala Asn Asp1
5 10 15548PRTArtificial SequenceSynthetic Construct 54Ser Ile Ile
Asn Phe Glu Lys Leu1 5559PRTArtificial SequenceSynthetic Construct
55Asn Leu Val Pro Met Val Ala Thr Val1 5
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